Health test for a broad spectrum of health problems

ABSTRACT

Provided herein are methods and devices for the detection of conditions or disorders by detecting altered levels of stress response pathway biomarkers. Also provided are methods and reagents for identifying panels of biontarkers associated with condition or disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 16/913,710 filed Jun. 26.2020, pending; which is a continuationapplication of U.S. application Ser. No. 16/421,089 filed May 23, 2019,now issued as U.S. Pat. No. 10,705,097; which is a continuationapplication of U.S. application Ser. No. 15/873,840 filed Jan. 17, 2018,now issued as U.S. Pat. No. 10,317,416; which is a continuationapplication of U.S. application Ser. No. 15/188,822 filed Jun. 21, 2016,now issued as U.S. Pat. No. 9,874,573; which is a continuation-in-partapplication of U.S. application Ser. No. 14/870,825 filed Sep. 30, 2015,now abandoned, which is a continuation application of U.S. applicationSer. No. 14/325,252 filed Jul. 7, 2014, now issued as U.S. Pat. No.9,176,149; which is a continuation application of U.S. application Ser.No. 13/122,130 filed Jun. 17, 2011, now issued as U.S. Pat. No.8.771,962; which is a 35 USC § 371 National Stage application ofinternational Application No. PCT/US2009/059438 filed Oct. 2, 2009, nowexpired; which claims the benefit under 35 USC § 119(e) to U.S.application Ser. No. 61/102,341 filed Oct. 2, 2008, now expired. Thedisclosure of each of the prior applications is considered part of andis incorporated by reference in the disclosure of this application.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to a device for detecting astress response in a sample, and more specifically to methods ofdetecting a stress biomarker in a sample.

Backcground Information

Wellness products and services are increasingly popular, particularly indeveloped countries. Many are rooted in the traditional medicine, e.g.,body care products, natural medicines, massage, acupuncture, sauna, spatreatments. New products and services include handheld devices for vitalsign measurements, tests for ‘good’ or ‘bad’ metabolites such asantioxidants or cholesterol, and treatments such as cold laser orhyperbaric oxygen. Wellness products and services are sold at retailstores, walk-in clinics, integrative medicine facilities, health spasand gyms and through electronic healthcare companies that also provideintegrated wellness services. Wellness products and services aretypically selected using generalized recommendations (e.g., age andgender based), subjective tests such as health questionnaires and thepain scale, vital signs (blood pressure), weight and metabolite tests(e.g., cholesterol or glucose). New test are needed for personalizedassessment of health, identification of the need for wellness productsand selecting the best match for the personal need. Optimally, new testswill provide an early warning of a health problem and indicate thenature of the problem, in addition, new tests showing specific healthbenefits of wellness products and services are needed, in addition tothe available anti-oxidant tests for nutritional supplements.

There is an urgent need for a new health test suitable for point-of-care(POC) settings. The test should be noninvasive and capable of detectingearly signs of deteriorating health status. To be POC-expedient, thetest should be rapid technically simple and inexpensive.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are provided. POCdevices useful for Stress Response Profiling (SRP) in saliva. In oneembodiment, the device is a hand-held digital device for mobile healthmonitoring (FIG. 6 ). The use of the device is intuitive (similar to adigital thermometer) and does not require training for use. The test isnoninvasive, rapid and inexpensive, and capable of detecting earlywarnings of health problems. The SRP device can be used for preventativePOC health screening, consumer-centric wellness care, routine clinicalcare, it can be used in emergency moms and trauma units, it can be usedby first responders, by individuals for chronic disease management, itcan be used in complementary and alternative medicine, militaryhealthcare.

In accordance with the present invention, there are provided devices fordetecting at least one stress response biomarker in a test sample. Suchdevices include a disposable module for uptake of a test sample andreagent storage, wherein the module contains reagents for assaying forat least one stress response biomarker; and a reusable module for signaldetection and result display; wherein the reusable module displays asignal that indicates the presence of the at least one stress responsehiomarker in the test sample. In some etribodiments, the signal providesa digital readout of a percentage above a baseline representing thepresence of the at least one stress response biomarker in the testsample. In certain embodiments, the signal provides a visual indicationrepresenting the presence of the at least one stress response biomarkerin the test sample. In one aspect, the visual indication is a colorindication.

The device may be used to test a biological sample. In some embodiments,the test sample is selected from the group containing breath air,saliva, urine, sweat, tears, blood, serum, stool, phlegm, bone marrow,cerebrospinal fluid, seminal fluid, vaginal fluid, amniotic fluid, skin,breast milk, tissue, plant sap, an egg, microbial body, cells suspensionor a combination thereof. In certain aspects the test sample is wholesaliva.

In certain embodiments, the device finther includes a means foraccessing a database, wherein the database provides a correlationbetween the presence of the at least one stress biomarker molecule inthe test sample and (i) the presence, absence, or severity, if present,of a particular disease state; or (ii) the likelihood that an organismfrom which the test sample was obtained will contract or be subject to aparticular disease state. In some embodiments, the device assays for atleast one stress response biomarker selected from the group consistingof Acidic Trehalase-like protein 1 (ATHL), Adrenocorticotropic hormone(ACTH), Aldose Reductase (ALR), ALG-2 interacting protein X (Alix),Annexin 5 (Annex), Apolipoprotein B mRNA editing enzyme APOBEC 3G (APO),Aquaporin 5 (AQP5), Betaine-GABA transporter 1 (BGT), Bone MarrowStromal Cell Antigen 2 (BST2, Tetherin), Caspase 3 (Casp3), Caspase 8(Casp8), CD63 (CC)h3, Tetraspanin, LAMP-3), CD9, Cyclin DI (Cyclin),Cyclooxygenase-2 (Cox-2), Cytochrome P450 2E1 (CYP450), Cytochrome P450Reductuse (CYPOR). Human beta defensing 2 (HBD2) Human beta defensin 3(HBD3), Human beta defensin 4 (HBD4), DICER, Epidermal growth factorreceptor (EGFR), Ferritin (Fer), Fos (Fos), Furin convertase (Furin,PACE). Glucocorticoid receptor (GR), Glucose regulated protein 58(Grp58), Glucose regulated protein 75 (Grp75, Mortalin), Giuthathione Stransferase pi (GS fp), Heat shock protein 27 (HSP27), Heat shockprotein 40 (HSP4O), Heat shock protein 60 (HSP60), Heat shock protein 70(HSP70), Heat shock protein 90 (HSP90), Heat shock protein transcriptionfactor 1 (FISF1), Heine oxygenase 1 (HO-1), Histone 3 methyltransferaseSUV39H (HAT), Histone deacetylase 1 (HDAC), Hyperosmotic glycerolresponse 1 (p38) (HOG), Hypoxia-induced factor alpha 1 WW1), Integrin B1(INTb), Interleukin-1 beta (1), IL-6 (IL-6), IL-8 (IL-8), IL-10 (IL-10),IL-12 beta (IL-12), Intracellular adhesion molecule-1 (ICAM CD54), Jun(Jun), Leptin, Leptin (obesity) receptor (ObR), Lysosome-associatedmembrane glycoprotein-1. (LAMP-2), MAP kinase p38 (p38), MAP kinaseMek-1, mitogen activated (MEK1), MAP kinase Mekk-i, stress activated(MEKK1), MAP kinase Jnk1/2, stress activated protein kinase (SAPK),Mammalian target of rapamycin (mTOR), Matrix metalloproteinase 9 (MMP9),Metallothionein (MT), Microtubule-associated protein light chain 3 β(MAP-LC3β, LC3), Mucin 1 (Muc1), Myeloperoxidase (MPO), Natriureticpeptide B (BNP), Natriuretic peptide receptor A (NPR), Neutrophilgelatinase-associated lipocalin 1 (NGAL). Neuropathy target esterase(NTE), Nitric oxide synthase, neuronal nNOS (NOS1), Nitric oxidesynthase inducible iNOS (NOS2), Nuclear factor of activated T cells 5(NFAT5, TonEBP), Ornithine decarboxylase (ODC), Osmotic stress protein94 (OSP94), Oxytocin receptor (OTR), Pro-opiomelanocortin/beta-endorphin(POMC), p53 tumor suppressor (p53), Peripheral benzodiazepine receptor(PBR), Salivary Agglutinin gp340 (SAG). Salivary alpha amylase (SAA),Secretory leukocyte protease inhibitor (SLP1), SodiumAnyo-inositolcotransporter (SMIT). Superoxide dismutase 1 Cu/Zn (SOD1), Superoxidedismutase 2 Mn (SOD2), Superoxide dismutase 3 Extracellular (SOD3),Substance P (SP), Substance P (Neurokinin 1) receptor (NKR), SerotoninReceptor 1A (SR1), Serotonin Receptor 2A (SR2). Taurin transporter(TauT), Tumor Growth Factor beta 1, 2, 3 (TGF), Toll-like receptor 2(TLR2), Toll-like receptor 3 (TLR3), Toll-like receptor 4 (TLR4),Toll-like receptor 7 (TLR7), Toll-like receptor 8 (TLR8), Trehalaseneutral (TRE), Ubiquitin (UB), Urotensin II (UT), Vascular adhesionmolecule-1 (VCAM1), Vascular endothelial growth factor C (VEGF-C), VEGFreceptor 1 (VEGFR-1, Flt-1) (VEGFR), Vasointestinal peptide (VIP),Vimentin (Vim) or a combination thereof. in one aspect, the deviceincludes at least one stress biomarker associated with dehydration. Inan additional aspect, dehydration is hypertonic dehydration, isotonicdehydration or hypotonic dehydration. In certain aspects, the at leastone stress biomarker associated with dehydration comprises AcidicTrehalase-like protein 1 (ATHL), Osmotic stress protein 94 (OSP94),Sodium/myo-inositol cotransporter (SMIT) or a combination thereof. Inanother aspect, the device includes at least one stress biomarkerassociated with AIDS progression (unsuppressed HIV) and/or acute HIV. Incertain aspects, the at least one stress biomarker associated with AIDSprogression (unsuppressed HIV) and/or acute HiV comprises Bone MarrowStromal. Cell Antigen 2 (BST2), Salivary Agglutinin gp340 (SAG),Vascular endothelial growth factor C (VEGF-C) or a combination thereof.In a further aspect, the device includes at least one stress biomarkerassociated with heart disease. In an additional aspect, heart disease isacute heart failure (AHF) with preserved ejection fraction (HF-pEF),acute heart failure (AHF) with restricted ejection fraction (HE-rEF) oratrial fibrillation (AFIB). In certain aspects, the at least one stressbiomarker associated with heart disease comprises Cyclooxygenase -2,Epidermal growth factor receptor, Leptin, MAP kinase Mek-1 or acombination thereof, In one aspect, the device includes at least onestress biomarker associated with traumatic brain injury (TBI). In anadditional aspect, traumatic brain injury is mild TBI (mTBI,concussion), severe TBI (sTBI) or neurocognitive disorder (NCD) due toTBI (NCDT). In certain aspects, the at least one stress biomarkerassociated with neurocognitive disorder (NCD) due to TBI (NCDT)comprises Cytochrorne P450 Reductase (CYPOR), Natriuretic peptidereceptor A (NPR), Oxytocin receptor (OTR) or a combination thereof. Inanother aspect, the device includes at least one stress biomarkerassociated with post-traumatic stress disorder (PTSD). In an additionalaspect, the device includes at least one stress biomarker associatedwith kidney disease, ineltiding chronic kidney disease (CKD) and acutekidney injury (AKI). In certain aspects, the at least one stressbiomarker associated with kidney disease comprises Annexin 5, Nuclearfactor of activated T cells 5, Osmotic stress protein 94 or acombination thereof.

In another embodiment of the invention, there are provided methods fordetecting a condition or disorder associated with a stress response in asubject. The methods include detecting an altered level of at least onebiomarkers in an stress response biomarker panel a sample comprisingsalivary cells from a stibject, as compared to a. corresponding samplefrom a normal subject, wherein the panel comprises at least twobiomarkers, and wherein further an alteration in the level of biomarkeris indicative of a stress response associated with the condition ordisorder, thereby detecting the condition or disorder in the subject. Inone embodiment, the sample is whole saliva. In particular embodiments,the at least one stress response biomarker is selected from the groupconsisting of Acidic Trehalase-like protein 1 (ATHL),Adrenocorticotropic hormone (ACTH), Aldose Reductase (ALR), ALG-2interacting protein X (Mix), Annexin 5 (Annex), Apolipoprotein B mRNAediting enzyme APOBEC 3G (APO). Aquaporin 5 (AQP5), Betaine-GABAtransporter 1 (BGT), Bone Marrow Stromal Cell Antigen 2 (BST2,Tetherin), Caspase 3 (Casp3), Caspase 8 (Casp8), CD63 (CD63,Tetraspanin, LAMP-3), CD9, Cyclin D1 (Cyclin), Cyclooxygenase-2 (Cox-2),Cytochrome P450 2E1 (CYP405), Cytochrome P450 Reductase (CYPOR), Humanbeta defensing 2 (HBD2,) Human beta defensin 3 (HBD3), Human betadefensin 4 (HBD4), DICER, Epidermal growth factor receptor (EGFR),Ferritin (Fer), Fos (Fos), Furin convertase (Furin, PACE),Glucocorticoid receptor (OR), Glucose regulated protein 58 (Grp58),Glucose regulated protein 75 (Grp75, Mortalin), Gluthathione Stransferase pi (OSTp), Heat shock protein 27 (HSP27), Heat shock.protein 40 (I-ISP40), Heat shock protein 60 (ISP60), Heat shock protein70 (HSP7O), Heat shock protein 90 (HSP90), Heat shock proteintranscription factor 1 (HSE1), Herne oxygenase 1 (HO-1), Histone 3methyltransferase SUV39H (HAT), Histone deacetylase 1 (IIDAC),Hyperosmotic glycerol response 1 (p38) (HOG), Hypoxia-induced factoralpha 1 (HIFI), Integrin 131 (INTb), Interleukin- I beta (IL-1), 1L-6(IL-6), IL-8 (IL-8). IL-10 (IL-10), IL-12 beta (IL-12), Intracellularadhesion molecule-1 (ICAM1 CD54), Jun (Jun), Leptin, Leptin (obesity)receptor (ObR). Lysosome-associated membrane glycoprotein- (LAMP-2), MAPkinase p38 (p38), MAP kinase Mek-1, mitogen activated (MEK1), MAP kinaseMekk-1, stress activated (MEKK1), MAP kinase ink1/2, stress activatedprotein kinase (SAPK), Mammalian target of rapamycin (mTOR), Matrixmetalloproteinase 9 (MMP9), Metallothionein (MT), Microtubule-associatedprotein light chain 3 β (MAP-LCβ, LC3), Mucin 1 (Muc1), Myeloperoxidase(MPG). natriuretic peptide B (BNP), Natriuretic peptide receptor A(NPR), Neutrophil gelatinase-associated lipoealin 1 (NGAL), Neuropathytarget esterase (NTE), Nitric oxide synthase, neuronal nNOS (NOS1),Nitric oxide synthase, inducible iNOS (NOS2), Nuclear factor ofactivated T cells 5 (NEAT5, TonEBP), Ornithine decarboxylase (ODC),Osmotic stress protein 94 (OSP94), Oxytocin receptor (OTR),Pro-opiomelanocortinibeta-endorphin (POMC), p53 tumor suppressor (p53),Peripheral benzodiazepine receptor (PBR), Salivary Agglutinin gp340(SAG), Salivary alpha, mylase (SAA), Secretory leukocyte proteaseinhibitor (SLPI), Sodium; myo-inositol cotransporter (SMIT), Superoxidedismutase 1 Cu/Zn (SODI), Superoxide dismutase 2 Mn (SOD2), Superoxidedismutase 3 Extracellular (SODS), Substance P (SP), Substance P(Neurokinin 1) receptor (NKR), Serotonin Receptor 1A (SR1), SerotoninReceptor 2A (SR2), Turin transporter (TauT), Tumor Growth Factor beta 1,2, 3 (TGF), Toll-like receptor 2 (TLR2), Toll-like receptor 3 (TLR3),Toil-like receptor 4 (TLR4), Toll-like receptor 7 (TLR7), Toll-likereceptor 8 (TLR8), Trehalase neutral (TRE), Ubiquitin (UB), Urotensin II(UT), Vascular adhesion molecule-1 (VCAM1), Vascular endothelial growthfactor C (VEGF-C), VEGF receptor 1 (VEGFR-1, Flt-1) (VEGFR),Vasointestinal peptide (VIP), Vimentin (Vim) or a combination thereof.In one aspect, the method includes at least one stress biomarkerassociated with dehydration. In an additional aspect, dehydration ishypertonic dehydration, isotonic dehydration or hypotonic dehydration.In certain aspects, the at least one stress biomarker associated withdehydration comprises Acidic Irehalase-like protein 1 (ATHL), Osmoticstress protein 94 (OSP94), Sodium/:myo-inositol cotransporter (SNIFF) ora combination thereof In another aspect, the method includes at leastone stress biomarker associated with AIDS progression (unsuppressed HIV)and/or acute HIV. In certain aspects, the at least one stress biomarkerassociated with AIDS progression (unsuppressed HIV) and/or acute HIVcomprises Bone Marrow Stromal Cell Antigen 2 (BST2), Salivary Agglutiningp340 (SAG), Vascular endothelial growth factor C (VEGF-C) or acombination thereof. In a further aspect, the method includes at leastone stress biomarker associated with heart disease. In an additionalaspect, heart disease is acute heart failure (AHF) with preservedejection fraction (HF-pEF), acute heart failure (AHF) with restrictedejection fraction (HF-rEF) or atrial (AFIB). In certain aspects, the atleast one stress biomarker associated with heart disease comprisesCyclooxygenase 2 (Cox-2). Epidermal growth factor receptor (EGFR),Leptin, MAP kinase Mek-1 or a combination thereof In one aspect, themethod includes at least one stress biomarker associated with traumaticbrain injury (FBI), in an additional aspect, traumatic brain injury ismild TBI (mTBI, concussion), severe TBI (sTBI) or neurocognitivedisorder (NCD) due to TBI (NCDT). In certain aspects, the at least onestress biomarker associated with neurocognitive disorder (NCD) due toTBI (NCDT) comprises Cytochrome P450 Reductase (CYPOR), Natriureticpeptide receptor A (NPR), Oxytocin receptor (OTR) or a combinationthereof. In another aspect, the method includes at least one stressbiomarker associated with post-traumatic stress disorder (PTSD). In anadditional aspect, the method includes at least one stress biomarkerassociated with kidney disease, including chronic kidney disease (CKDand acute kidney injury (AKI). In certain aspects, the at least onestress biomarker associated with kidney disease comprises Annexin 5(Annex), Nuclear factor of activated T cells 5 (NFAT5, TonEBP), Osmoticstress protein 94 (OSP94) or a combination thereof.

In some embodiments, the levels of the at least one biomarker aredetected by analysis of biomarker protein or nucleic acid in the samplecomprising the salivary cells. In particular embodiments, the analysisof biomarker protein includes detection with an antibody. In one aspect,the salivary cells are lysed prior to analysis with the antibody, Theanalysis may be conducted by ELISA or other antibody detection methodsknown in the art, in certain embodiments, the levels of the at least onebiomarker are assayed using a device of the invention. In someembodiments, the sample contaMing the salivary cells is analyzed onmicroscope slide. In one embodiment, the sample is whole saliva.

In other embodiments, the analysis of biomarker nucleic acid comprisesisolation of salivary cell nucleic acid In one aspect, the biomarkernucleic acid is detected in the isolated salivary cell nucleic acid bynucleic acid hybridization or PCR amplification.

In another embodiment of the invention there are provided methods ofprocessing a salivary cell sample for biomarker analysis. Such methodsinclude applying a sample of saliva or salivary cells to a substrate;fixing the cells; incubating the cells in low pH citrate buffer at 37°C.; contacting the cells with serum; applying a primary antibody foreach of biomarker of a biomarker panel; and detecting the binding of theprimary antibody using a secondary antibody having a detectable label,wherein the label is detected optically using a computerized imageanalysis. In certain embodiments, the salivary cells are collected usingan oral brush. In some embodiments, the biomarker panel comprises atleast one biomarker selected from the group consisting of AcidicTrehalase-like protein 1 (ATHL), Adrenocorticotropic hormone (ACTH),Aldose Reductase (ALR), ALG-2 interacting protein X (Alix), Annexin 5(Annex), Apolipoprotein B mRNA editing enzyme APOBEC 3G (APO), Aquaporin5 (AQP5), Betaine-GABA transporter 1 (BGT), Bone Marrow Stromal CellAntigen 2 (BST2, Tetherin), Caspase 3 (Casp3), Caspase 8 (Casp8), CD63(CD63, Tetraspanin, LAMP-3), CD9, Cyclin D1 (Cyclin), Cyclooxygenase -2(Cox-2), Cytochrome P450 2E1 (CY P450), Cytochrome P450 Reductase(CYPOR), Human beta defensing 2 (HBD2,) Human beta defensin 3 (HBD3),litmlan beta defensin 4 (HBD4), DICER, Epidermal growth factor receptor(EGER), Ferritin (Fer), Fos (Fos), Furin convertase (Furin, PACE),Glucocorticoid receptor (OR), Glucose regulated protein 58 (Grp58),Glucose regulated protein 75 (Orp75, Mortalin), Gluthathione Stransferase pi (GSTp), Heat shock protein 27 (HSP27), Heat shock protein40 (HSP40). Heat shock protein 60 (HSP60), Heat shock protein 70(HSP70), Heat shock protein 90 (HSP90), Heat shock protein transcriptionfactor 1 (HSF1), Herne oxygenase 1 Histone 3 methyltransferase SUV39H(HAT), Histone deacetylase 1 (HDAC), Hyperosmotic glycerol response 1(p38) (HOG), Hypoxia-induced factor alpha 1 (HIF1), Integrin B1 (INTb),Interleukin-1 beta (IL-1), IL-6 (IL-6), IL-8 (IL-8), IL-10 (IL-10),IL-12 beta (IL-12), Intracellular adhesion molecule-I (ICAM1, CD54), Jun(Jun), Leptin, Leptin (obesity) receptor (ObR), Lysosome-associatedmembrane glycoprotein-1 (LAMP-2), MAP kinase p38 (p38), MAP kinaseMek-1, mitogen activated (MEK1), MAP kinase. Mekk-1, stress activated(MEKK1), MAP kinase Jnk1/2, stress activated protein kinase (SAPK),Mammalian target of rapamycin (mTOR), Matrix metalloproteinase 9 (MMP9),Metallothionein (MT), Microtubule-associated protein light chain 3 β(MAP-LC3β, LC3), Mucin 1 (Muc1), Myeloperoxidase (MPO), Natriureticpeptide B (BNP), Natriuretic peptide receptor A (NPR), Neutrophilgelatinase-associated lipocalin 1 (NGAL), Neuropathy target esterase(NTE), Nitric oxide synthase, neuronal nNOS (NOS1). Nitric oxidesynthase, inducible iNOS (NOS2), Nuclear factor of activated T cells 5(NFAT5, TonEBP), Ornithine decarboxylase (ODC),. Osmotic stress protein94 (OSP94), Oxytocin receptor (OTR),Pro-opiornelanocortinfbeta-endorphin (POMC), p53 tumor suppressor (p53),Peripheral benzodiazepine receptor (PBR), Salivary Agglutinin gp340(SAG), Salivary alpha amylase (SAA), Secretory leukocyte proteaseinhibitor (SLPI), Soditiniimyo-inositol cotransporter (SMIT), Superoxidedismutase 1 Cu/Zn (SOD1), Superoxide dismutase 2 Mn (SOD2), Superoxidedismutase 3 Extracellular (SOD3), Substance P (SP), Substance P(Neurokinin 1) receptor (NKR), Serotonin Receptor 1A (SRI), SerotoninReceptor 2A (SR2), Taurin transporter (TauT), Tumor Growth Factor beta1, 2, 3 (TGF), Toll-like receptor 2 (TLR2), Toll-like receptor 3 (TLR3),Toll-like receptor 4 (TLR4), Toll-like receptor 7 (TLR7), Toll-likereceptor 8 (TLR8), Trehalase neutral (TRE), Ubiquitin (UB), Urotensin II(UT), Vascular adhesion molecule-1 (VCAM1), Vascular endothelial growthfactor C (VEGF-C), VEGF receptor 1 (VEGFR-1. Flt-1) (VEGFR),Vasointestinal peptide (VIP), Vimentin (Vim) or a combination thereof.

In another embodiment of the invention, there are provided methods forconstructing a biomarker panel for detecting a stress response in acultured cell. The method includes detecting the level of one or morebiomarkers from a panel of biomarkers in cultured cells subjected to atreatment that induces cellular stress; and comparing the level of thebiomarkers from the treated cells to the level of the biomarker from acorresponding sample of cultured cells that have not been subjected tothe treatment that induces cellular stress, wherein biomarkers having adifference level in the treated cells as compared to the untreated cellsare included in an SR biomarker panel for a stress response. Inparticular embodiments, the treatment that induces cellular stress is astressor selected from the group consisting of heat shock, freeze/thawcycling, hypersalinity, dehydration, and oxidative stress. In someembodiments, the cultured cells are salivary cells, peripheral bloodmononuclear cells, or cells from organ cultures of tonsil, skin, gut orlung. In particular embodiments, the cells are animal cells. In oneaspect, the cells are human cells.

In one embodiment, the present invention provides a method for detectinga condition or disorder associated with a stress response in a subjectcomprising detecting an altered level of at least one biomarkers in anstress response biomarker panel in a sample comprising salivary cellsfrom a subject, as compared to a corresponding sample from a normalsubject, wherein the panel comprises at least two biomarkers, andwherein further an alteration in the level of biomarker is indicative ofa stress response associated with the condition or disorder, wherein thealtered levels of the at least one or more biomarkers are detected usinga device of claim 1, thereby detecting the condition or disorder in thesubject.

In one aspect, the at least one biomarker selected from the groupconsisting of Acidic Trehalase-like protein 1 (ATHL),Adrenocorticotropic hormone (ACTH), Aldose Reductase (ALR), ALG-2interacting protein X (Mix), Annexin 5 (Annex), Apolipoprotein B mRNAediting enzyme APOBEC 3G (APO), Aquaporin S (AQP5), Betaine-GABAtransporter 1 (BGT), Bone Marrow Stromal. Cell Antigen 2 (BST2,Tetherin), Caspase 3 (Casp3), Caspase 8 (Casp8), CD63 (CD63.1etraspanin,LAMP-3), CD9, Cyclin D1 (Cydin), Cyclooxygenase -2 (Cox-2), CytochromeP450 2E1 ((YP450), Cytochrome P450 Reductase (CYPOR), Human betadefensing 2 (HBD2,) Human beta defensin 3 (HBD3), Human beta defensin 4(HBD4), DICER, Epidermal growth factor receptor (EGFR), Ferritin (Fer),Fos (Fos), Furin convertase (Furin, PACE), Glucocorticoid receptor (GR),Glucose regulated protein 58 (Grp58), Glucose regulated protein 75(Grp75, Mortalin), Gluthathione S transferase pi (GSTp), Heat shockprotein 27 (HSP27), Heat shock protein 40 (HSP40), Heat shock protein 60(HSP60), Heat shock protein 70 (HSP70), Heat shock protein 90 (HSP90),Heat shock protein transcription. factor 1 (HSF1), Hemee oxygenase 1(HO-1), Histone 3 methyltransferase SUV3914 (HAT), Histone deacetylase 1(HDAC), Hyperosmotic glycerol response 1 (p38) (HOG), Hypoxia-inducedfactor alpha 1 (HW1). Integrin B (INTb). Interleukin- beta (IL-1), IL-6(IL-6), IL-8 (IL-8), IL-10 (IL-12 beta (IL-12), hrtracellular adhesionmolecule-1 (ICAM1, CD54), Jun (Jun), Leptin, Leptin (obesity) receptor(ObR), Lysosome-associated membrane g1yeoprotein-1 (LAMP-2), MAP kinasep38 (p38), MAP kinase Mek-1, mitogen activated (MEK1), MAP LinageMekk-1, stress activated (MEKK1), MAP kinase Jnk1/2, stress activatedprotein kinase (SAPK). Mammalian target of rapamycin (mTOR), Matrixmetalloproteinase 9 (MMP9), Metallothionein (MT), Microtubule-associatedprotein light chain 3 β (MAP-LC3β, LC3), Mucin 1 (Muc1), Myeloperoxidase(MPO), Natriuretic peptide B (BNP), Natriuretic peptide receptor A(NPR), Neutrophil gelatinase-associated lipocalin 1 (NGAL), Neuropathytarget esterase (NTE), Nitric oxide synthase, neuronal nNOS (NOS1),Nitric oxide synthase, inducible iNOS (NOS2), Nuclear factor ofactivated T cells 5 (NFAT5, TonF BP), Ornithine decarboxylase (ODC),Osmotic stress protein 94 (OSP94), Oxytocin receptor (OTR),Pro-opiomelanocortinibeta-endorphin (POMC), p53 tumor suppressor (p53),Peripheral benzodiazepine receptor (PBR), Salivary Agglutinin gp340(SAG), Salivary alpha amylase (SAA), Secretory leukocyte proteaseinhibitor (SLPI), Sodiumftnyo-inositol cotransporter (SMIT), Superoxidedismutase 1 CU/Zn (SOD1), Superoxide dismutase 2 Mn (SOD2), Superoxidedismutase 3 Extracellular (SOD3). Substance P (SP), Substance P(Neurokinin 1) receptor (NKR), Serotonin Receptor 1A (SR1), SerotoninReceptor 2A (SR2), Taurin transporter (TauT), Tumor Growth Factor beta1, 2, 3 (TGF), Toll-like receptor 2 (TLR2), Toll-like receptor 3 (TLR3).Toll-like receptor 4 (TLR4), Toll-like receptor 7 (TLR7), Toll-likereceptor 8 (TLR8), Trehalase neutral (TRE), B), Ubiquitin II (UT),Vascular adhesion molecule-1 (VCAM1), Vascular endothelial growth factorC (VEGF-C), VEGF receptor 1 (VEGFR-1, Flt-1) (VEGFR), Vasointestinalpeptide (VIP), Vimentin (Vim) or a combination thereof.

In another aspect, the condition or disorder associated with a stressresponse is dehydration, AIDS progression (unsuppressed HIV), acute HIV,traumatic brain injury (TBI), post-traumatic stress disorder, heartdisease or kidney disease. In an additional aspect, dehydration ishypertonic dehydration, isotonic dehydration or hypotonic dehydration.In certain aspects, the at least one stress biomarker associated withdehydration comprises Acidic Trehalase-like protein 1 (ATHL), Osmoticstress protein 94 (OSP94), Sodium/myo-inositol cotransporter (SMIT) or acombination thereof In a further aspect, the at least one stressbiomarker associated with AIDS progression (unsuppressed WV) and/oracute HIV comprises Bone Marrow Stromal Cell Antigen 2 (BST2), SalivaryAgglutinin gp340 (SAG), Vascular endothelial growth factor C (VEGF-C) ora combination thereof. in one aspect, heart disease is acute heartfailure (AHF) with preserved ejection fraction (HF-pEF), acute heartfailure (AHF) with restricted ejection fraction (HF-rEF) or atrialfibrillation (AFIB). In another aspect the at least one stress biomarkerassociated with heart disease comprises Cyclooxygenase -2 (Cox-2),Epidermal growth factor receptor (EGFR), Leptin, MAP kinase Mek-1 or acombination thereof. In an additional aspect, traumatic brain injury ismild TBI (mIBI, concussion), severe TBI (sTBI) or neurocognitivedisorder (NCD) due to TBI (NCDT). In a further aspect, the at least onestress biomarker associated with neurocognitive disorder (NCD) due toTBI (NCDT) comprises Cytochrome P450 Reductase (CYPOR), Natriureticpeptide receptor A (NPR). Oxytocin receptor (OTR) or a combinationthereof. In another aspect, kidney disease is chronic kidney disease(CKD) or acute kidney injury (AKI). In an additional aspect, the atleast one stress biomarker associated with kidney disease comprisesAnnexin 5 (Annex), Nuclear factor of activated T cells 5 (NFAT5,TonEBP), Osmotic stress protein 94 (OSP94) or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustration of device for collecting saliva samples.

FIG. 2 shows a plot of the levels of stress response (SR) biomarkers insalivary cells treated with different environmental stressors. SRbiomarkers were detected using enzymatic immunochemical staining assaywith mouse and rabbit antibodies and permanent color label. The stainingintensity was measured in relative optical density units using imageanalysis. The y axis shows the average staining intensity for 40 SRbiomarkers. Error bars show standard deviations of the average stainingintensity. N pool, untreated salivary cells from three donors. Culturedcells from the N pool were treated by desiccation (T1), hypersalinity(T2) or heat shock (T3). S pool, cells combined after treatment of the Npool by desiccation, hypersalinity, heat shock, oxidative stress andfreeze/thaw shock.

FIGS. 3A-3H show cellular stress in saliva induced by treatment o o byphysiological stress in vivo. 40 SR biomarkers were detected in salivarycells using immtmochemical staining with a color label. In cell images,the color is shown as stippling. FIG. 3A. N pool, normal saliva from 3donors. S pool, cells combined after treatment by desiccation,hypersalinity, heat shock, oxidative stress and freeze/thaw shock. FIG.3C. Salivary cells from a healthy donor. FIG. 3D. Saliva from the samedonor during post-traumatic physiological stress. FIG. 3E. Saliva afterexposure to cellular stress. FIG. 3F. Saliva after exposure to cellularstress. FIG. 3G. EMA, positive control staining of a cytoplasmic proteinin salivary epithelial cells. FIG. 3H. NC, a negative staining control.

FIG. 4 shows a plot of shows levels of individual SR biomarkers beforeand after treatment with environmental stressors. 52 SR biomarkers weremeasured using immunochemical staining and image analysis. The y axisshows the average staining intensity for the 52 biomarkers. N pool,untreated salivary cells from three donors. S pool, cells combined aftertreatment by desiccation, hypersalinity, heat shock, oxidative stressand freezefthaw shock.

FIG. 5 shows expression profiles of SR biomarkers induced by treatmentof salivary cells with different stressors, 52 SR biomarkers weremeasured using immunochemical staining and image analysis. Themeasurements were analyzed using hierarchic clustering to depictrelatedness between profiles. The color scheme indicates biomarkerlevels. The lowest level is white, increased levels are gray to black.Similar profiles are in clusters with short dendrogram branches. N,untreated salivary cells from three donors. S, cells combined aftertreatment by desiccation, hypersalinity, heat shock, oxidative stressand freeze/thaw shock. T1a and T1b, desiccation, two subjects. T2,hypersalinity. T3, heat shock.

FIG. 6 shows an illustration of a rapid, hand-held test device tiersaliva biomarkers. The device consists of a disposable cartridge foruptake of saliva sample and reagent storage, and a reusable reader forsignal detection and result display. The result indicates the presenceof a saliva biomarker. The digital display window identifies results asnormal tdisplay reads “OK”), moderately abnormal (display reads“CAUTION”) or highly abnormal (display reads “DANGER”).

FIGS. 7A-7B. FIG. 7A shows images of multi-SRP staining of saliva FIG.7B shows a plot of the SRP score calculated as the ratio between theaverage staining intensity across 900 saliva cells, and the maximumstaining intensity value for saliva cells.

FIGS. 8A-8C show the pathway signature for dehydration. FIG. 8A. Acute4% hypertonic dehydration. FIG. 8B. Chronic (12 hrs) 4% hypertonicdehydration. FIG. 8C. Dehydration and concussion. The pathways are1—Redox, 2—Osmotic stress, 3—Cellular detox. 4—Chaperoning, 5—DNA,6—Adhesion, 7—Cell cycle, 8—Apoptosis, 9—Signaling, 10—Immunity,11—Microbiome.

FIGS. 9A-9B show the validation of Mucin 1 IHC assay for whole saliva.FIG. 9A. Images of 1× to 40× concentrated saliva stained for Mucin 1(magnification ×200). FIG. 9B. Standard calibration curve for the Mucin1IHC assay.

FIG. 10 shows Western blot analysis of whole saliva. Calibration curvewas constructed using 7 serial dilutions of recombinant Hsp27(triangles): 100 pg-4 ng/lane (100 ng-4 ug/ml). 4 dilutions of wholesaliva (wen circles) used to determine the Hsp27 protein concentrationin the whole saliva.

FIG. 11 is a table showing candidate biomarkers of dehydration.Diagnostic accuracy was calculated as the percent AUC value from ROCcurve analysis. Diagnostic accuracy values ≥80% are bolded. DEH,dehydration. REH, rehydration. EUH, euhydration.

FIGS. 12A-12B show the fold increase in normalized biomarker levelsduring dehydration relative to euhydrated baseline. FIG. 12A. Thedehydration and SRP pathways associated with 80 SRP biomarkers. FIG.12B. SRP pathways involved in acute dehydration, chronic dehydration anddehydration and concussion.

FIGS. 13A-13C show the diagnostic accuracy, specificity andsensitivitywas determined tier different types and levels ofdehydration. FIG. 13A. Hypertonic dehydration (2% and 4%) isotonicdehydration, comb dehydration (hypertonic and isotonic), rehydration andcontrol. FIG. 13B. Gender effect and daily and diurnal variability. FIG.13C. Comparison of diagnostic accuracy between biomarkers and standardindicators.

FIGS. 14A-14D show the correlation between final H.SM biomarkers andstandard hydration iitclicators. Panel score was correlated withmeasurements of FIG. 14A. Body Mass Loss (BML%), FIG. 148 , PlasmaSodium (Sodium), FIG. 14C. Plasma Osmolality (Posen) and FIG. 14D. UrineSpecific Gravity (USG). Key: E, euhydration. D, dehydration. R,rehydration.

FIGS. 15A-15C show the pathway signature of IV infection. FIG. 15A. TheHIV/AIDS and SRP pathways associated with the 100 SRP biomarkers. FIG.15B. Suppressed HIV and unsuppressed FIG. 15C. Acute HIV and STD. Thepathways are 1) Redox; 2) Detox; 3) Chaperoning; 4) DNA; 5) Adhesion; 6)Cell cycle/energy; 7) Apoptosis; 8) Signaling; 9) Immunity and 10)Microbiome,

FIG. 16 shows the markers for unsuppressed HIV.

FIGS. 17A-17I show the pathway signature of Neuro-Cognitive Disorder dueto TBI. (NCDT) and other diseases. FIG. 17A. The TBI/NCDT pathways andSRP pathways and 100 SRP biomarkers, Normalized. Pathway activationindex (0-10) was calculated from biomarker data using a patentedalgorithm. FIG. 1713 . Acute TBI. FIG. 17C. Acute dehydration, FIG. 17D.HF-pEF (heart failure with preserved ejection fraction). FIG. 17E. AcuteHIV, FIG. 17F. Chronic NCD due to TBI. FIG. 17G. Chronic dehydration.FIG. 17H. HF-rEF (heart failure with reduced ejection fraction). FIG.17I. Chronic HIV/AIDS. The arrow indicates the top activated pathway ineach disease. The SRP pathways: 1—Oxidative stress, 2—Detoxification,3—Chaperoning, 4—DNA, 5—Adhesion/Cytoskeleton, 6—Cell cycle,7—Apoptosis, 8—Signaling, 9—Immunity, 10—Microbiome.

FIGS. 18A-18B are a table of Stress Response Profiling (SRP) biomarkersand the associated SRP pathways. FIG. 18A. Specific biomarkersassociated with pathways. The pathways are 1) Redox stress response; 2)Cellular detoxification; 3) Protein chaperoning; 4) DNA repair andmodification; 5) Cell adhesion, cytoskeleton, exosomes; 6) Cell cycle &energy metabolism; 7) Apoptosis and autophagy; 8) Neuroendocrine.signaling; 9) Innate and specific immunity; 10) Microhiome stressresponse and 11) Microbiome stress response. FIG. 18B. SRP pathways anda heat map for SRP biomarkers.

FIGS. 19A-19B are tables showing candidate saliva biomarkers fordimmostics of heart disease imd kidney disease. FIG. 19A. Salivabiomarkers for diagnostics of AHF, HF-pEF, HF-rEF and and AFIB. FIG.19B. Saliva biomarkers for diagnostics of AKI and CKD. Diagnosticaccuracy was calculated as the percent AUC value front ROC curveanalysis. Key: AHF, Acute Heart Failure; AFIB, Atrial Fibrillation; AKI,Acute Kidney Injury; CDK, Chronic Kidney Disease. EF, Ejection Fraction;HF-pEF, Heart Failure with preserved EF; HF--rEF, Heart Failure withrestricted EF.

FIGS. 20A-20C show candidate saliva bio.markers for diagnostics of acutemTBI (concussion) in 2 independent human studies. FIG. 20A. Figureshowing OSP94 and Vimentin biomarkers are strongly upregulated in wholesaliva from inTBI patients relative to healthy controls in Study 1. FIG.20B. Table showing fold increase in 75 SRP biomarkers in mTBI salivarelative to healthy controls in Study 1. FIG. 20C. Table showing foldincrease in 50 SRP biomarkers in triTBI saliva relative to healthycontrols in Study 2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to devices and methods tbr detecting astress response in a sample. The present invention also relates todetermining and detecting levels of biomarkers in a sample.

Biological responses to stressors involve hundreds of highly integratedmolecular pathways. However, to practically analyze chronic stress, asmall number of “universal pathways” have been identified thatreproducibly respond to most stressors in most organisms, and inparticular, essentially all vertebrates. Functional activation of theseuniversal pathways by stressors generates reproducible patterns of datathat can be monitored to analyze the characteristics and effects ofchronic stress.

The methods described herein are referred to as “stress responseprofiling” or “SR profiling,” because they relate to the measurement ofthe levels of multiple SR hiomarkers by performing SR hiomarker assays,where the SR biomarkers are associated with multiple stress responsepathways that are reproducibly activated by chronic stress (i.e., theuniversal SR pathways.) The results of such multi-dimensional SRbiomarker assays can be used to construct a “profile” (i.e. a pattern ofdata, which is also referred to in the industry as a “signature” or a“fingerprint”) that is characteristic of the type of stress, theorganism and/or the sample type.

Stressors can trigger persistent perturbations of homeostasis, i.e.,they cause chronic stress. Biological responses to chronic stress (alsoreferred to as “adaptive stress responses”) can be categorized in termsof the SR pathways they activate, which are further characterized interms of the SR biomarkers associated with these pathways. Thus, SRprofiling of either or both the SR pathway activation or the SRbiomarker levels resulting from such activation can be utilized toprovide molecular signatures of biological responses to stressors thatthreaten health, such as stressors that cause chronic s SR profiling istheretbre useful, in part, to predict increased risk of disease.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of ordinary skillin the art to which this invention belongs. One of ordinary skill in theart will also appreciate that any methods and materials similar orequivalent to those described herein can also be used to practice ortest this invention.

With respect to ranges of values, the invention encompasses eachintervening value the upper and lower limits of the range to at least atenth of the lower limit's unit, unless the context clearly indicatesotherwise. Moreover, the invention encompasses any other statedii:itervening values and ranges including either or both of the upperand lower limits of the range, unless specifically excluded from thestated range.

As used in this specification and in the appended claims, the singu.larforms include the plural forms. For example the terms “a,” “an,” and“the” include plural references unless the content clearly dictatesotherwise. Additionally, the term “at least” preceding a series ofelements is to be understood as referring to every element in theseries. The inventions illustratively described herein can suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, thetenns “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no ii:itention in the use of such termsand expressions of excluding any equivalents of the future shown anddescribed or any portion thereof, and it is recognized that variousmodifications are possible within the scope of the invention claimed.

-   -   1. SRP Technology

Stress Response Profiling (SRP) is a recently developed technology thatuses molecular biomarkers for multiparametric measurements ofphysiological stress responses. The SRP measurement serves as a novelvital sign. SRP is applicable to a broad spectrum of health threatsincluding environmental stressors, metabolic stressors, psychologicaltrauma, injuries and diseases. SRP measurements quantify physiologicalstress and also discriminate between different types of healthdisorders, SRP biomarkers monitor ten principal homeostatic processes(Table 1).

TABLE 1 SR Biomarkers Monitor Ten Principal Cellular Stress Responses SRbiomarkers SR 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2223 24 25  1 + + + + + + + + + + + + + +  2 + + + + + + + + + 3 + + + + + + + + +  4 + + + + + +  5 + + + + + + + + 6 + + + + + + + + + + + + + + + + + + + + + + + + 7 + + + + + + + + + + + + +  8 + + + + + + + + + + + + + + + + 9 + + + + + + + + + + + + + + + + + + + + + + 10 + + + + SR 26 27 28 2930 31 32 33 34 35 36 37 38 39 40  1 + + + + + +  2 +  3  4 + + + + 5 + + + + + +  6 + + + + + + + + + + + + + + +  7 + + + + + + + + + + + 8 + + + + + + + + + + +  9 + + + + + + + + + + + + + 10 + + + + + + SRPbiomarkers 1-40: beta-endorphin, caspase 8, cyclin D, Cox-2, CYP450,cytochrome c, EGFR, ferritin, glucocorticoid receptor, Grp58, Grp75,GSTp, Hsp25/27, Hsp40, Hsp60, Hsp70, Hsp90, HSF1, HO-1, IL-1 beta, IL-6,IL-8, IL-10, laminin, leptin receptor, metallothionein, Mekk-1, Mek-1,NADPH-CYP450 reductase, iNOS, Fos, Jun, serotonin receptor, serotonin,Substance P, SOD Mn, SOD Cu/Zn, TGFbeta, p53, vasoactive intestinalpeptide. SR, stress responses 1-10; redox control, cellulardetoxification Phase I and II, chaperoning, DNA repair, cellularadhesion and motility, cell growth and energy metabolism, apoptosis,neuro-endocrine signaling, immunological activation, microbialactivation.

As used herein, the term “homeostasis” is a biological process thatmaintains the health of organisms.

As used herein, the term “persistent homeostatic perturbation” is to beunderstood as a homeostatic change that has an adverse effect on thehealth of organisms. It is another way of referring to “chronic stress”or simply “stressed” which should be understood to mean a persistentperturbation of homeostasis and encompassing all forms of chromecellular stress and chronic physiological stress.

As used herein, the term “stressor” is to be understood as all thrillsof agents or conditions that give rise to stress. Stressors according tothe present invention include agents and conditions that are in theouter environment of organisms such as the air temperature as well asagents and conditions that are in the inner environment of omanisms suchas a disease.

As used herein, the term “adaptive stress response” or simply “stressresponse” is to be understood as a homeostatic process that provides acountermeasure to stress.

As used herein, the term “stress response pathway” is to be understoodas the form of the stress response that has a specific function in theorganism, such as DNA repair. Stress response pathways are embodied inexpressed molecules (i.e., SR biomarkers.)

As used herein, the term “universal stress response pathway” or simply“SR pathway” is to be understood as a tbrm of stress response to moststressors, in most organisms. Functional activation of these. SRpathways generates reproducible patterns of expressed molecules.

As used herein, the term “SR biomarker” is to be understood as anexpressed molecule known to be or suspected of being associated withactivation of a SR pathway.

As used herein, the term “SR biomarker profile” is a multi-dimensionalpattern of data whose components are at least two SR biomarker scoresfor individual SR biomarkers across a SR biomarker panel.

As used herein, the terms “SR pathway profile” and “SRP” are amulti-dimensional pattern of data representing at least two SR pathways.The components are functions of SR biomarker scores related to theindividual SR pathways. The functions yield one-dimensional data pointsthat provide simple-to-use indices of activation levels for theindividual pathways.

As used herein, the term “stress response profiling” refers toconstructing either or both SR pathway profiles or SR biomarker profilesfrom SR biomarker assays.

As used herein, the term “SR biomarker panel” is to be understood as atleast two SR biomarkers that as a group provide enhanced informationabout stress responses than single SR biomarkers.

As used herein, the term “SR biomarker panel score” or “panel score” isto be understood as a one-dimensional data point calculated as theaverage of SR biomarker scores across a SR biomarker panel.

As used herein, the term “SR biomarker score” is to be understood as anormalized and optionally log-transformed measurement of a SR biomarker.

As used herein, the term “measurement” of a SR biomarker is to beunderstood as a quantitative or qualitative determination of the SRbiomarker's expression level in a sample from an organism.

As used herein, the term “individual SR biomarker assay” or “SRbiomarker assay” is to be understood as an assay of individual SRbiomarkers.

As used herein, the term “combined SR biomarker assay” is to beunderstood as an assay that yields measurements representative of thecombined expression levels for a panel of SR biomarkers.

The homeostatic processes monitored by SRP biomarkers regulate generalstress responses, basic body functions and physical vital signs (bodytemperature, heart rate FIR, blood pressure BP, respiratory rate). Thesehomeostatic processes include redox control, cellular detoxification,chaperoning, DNA repair, cellular adhesion and cell growth, apoptosis,neuron-endocrine signaling, immunity, microbial activation and osmoticstress.

Redox Control (1). This pathway regulates levels of reactive oxygen andnitrogen species (superoxide, nitric oxide, carbon monoxide) throughfree radical scavenging proteins such as superoxide disinutases. Freeradicals are essential cellular mediators but when in excess, they causecellular dysfunction through damaging lipids, proteins, DNA and membraneintegrity.

Cellular Detoxification (2). Cellular detoxification provides a defenseagainst chemical threats to cellular integrity. Phase I detoxificationis a cytochrome P450 driven process for metabolizing a wide variety ofendonnous metabolites (e.g., fatty acids, steroids) and foreignsubstances (drugs, alcohol, pesticides and hydrocarbons). Phase II isbased on the glutathione metabolism and provides cellular resistance tooxidants, hydrocarbons and heavy metals.

Chaperoning (3). Chaperones fold newly synthesized polypeptides anddenatured proteins and for prevent uncontrolled protein aggregation.Chaperoning involves hundreds of “client” proteins and therefore has akey role in multiple biological functions including cellular protection,metabolism, growth, the development of multicellular organisms andmolecular evolution. Excessive chaperoning facilitates disease byfolding “wrong” clients such as the diphtheria toxin or mutant p53 thatare cytotoxic or cause cancer.

DNA Repair (4). DNA damage is ubiquitous and therefore the stability ofthe genome is under a continuous surveillance by multiple DNA repairmechanisms. DNA lesions are produced during transcription andreplication, and by metabolic and immunity by-products (e.g., freeradicals produced during aerobic respiration and by immune cells killingbacteria). DNA can be also damaged by environmental mutagens such asoxidants, heavy metals, radiation and viruses. The DNA repair pathwayregulates multiple stages and mechanisms of DNA repair, and is closelylinked with cell cycle control and apoptosis.

Cellular Adhesion and Motility (5). This pathway monitors cellularinteractions with the extracellular matrix and also changes incytoskeletal matrix such as centrioles, kinetosomes and othermicrotubule organizing centers. These processes are essential forcellular survival, gowth, metabolism and motility, and also for theformation of microbial biofilms and microbial-host interactions.

Cell Growth (6). In multicellular organisms, cell cycle progression isstrongly regulated during the development and modulated by growthfactors (mitogens), disease and environmental stress. In mature tissues,most cells do not divide. Cycling cells in tissues are typically somaticstem cells involved in normal tissue turnover (e.g., the germinal layerof the skin). Cell cycling is typically arrested in starved cells and incells with DNA or mitochondrial damage. Increased cell growth occursduring immune responses, wound healing and regeneration of tissuesdamaged by environmental stress, toxins, disease or infection.Uncontrolled, excessive cell growth is found in cancer.

Cell Death (7). The programmed cell death (apoptosis) “recycles”cellular components and prevents the release of toxins from dying cells,as happens during necrotic cell death. In animal tissues, apoptosis isincreased in areas of tissue remodeling and wound healing, and duringaging. During a disease, apoptosis can be increased within the diseasedtissue (e.g., psoriatic skin lesions) and/or in remote tissues andbiotluids HIV Tat protein is a soluble mediator that triggers apoptosisin uninfected lymphocytes). Apoptosis can be also triggered byenvironmental stressors that cause mitochondrial damage (e.g., oxidativestress and uv

Neuro-Endocrine Signaling (8). This pathway is crucial for regulatingphysiological homeostasis and behavioral regulation in animals includingsimple invertebrates. It involves a large number of mediators (hormones,neuropeptides. neurotransmitters) and cellular receptors produced byspecialized tissues (glands and neural tissues), and also locally inperipheral tissues (e.g., skin and gut). In vertebrates, two signalingmechanisms provide initial responses to stress: the limbichypothalamic-pituitary-adrenal (LHPA) axis that involves glucocorticoids(e.g., Cortisol) and the sympathetic nervous system activation viacatecholamines. However, chronic stress also activates signaling of painand anxiety, energy balance, metabolism, respiration, circulation andreproduction. Neuro-endocrine and immune signaling are integratedthrough common mediators and provide coordinated responses toenvironmental stress and disease.

Immunity (9). Inununity provides a systemic defense against biologicalthreats to organism's integrity such as injuries, tumors anddisease-causing microorganisms. Innate immunity provides a nonspecificdefense through soluble mediators (e.g., chemokines, agglutinins) andspecialized cells (e.g., macrophages) that circulate through theorganism and inactivate parasitic microorganisms, engulf apoptotic celldebris and kill infected and tumor cells. Innate immunity is found inprotists, animals and plants, Vertebrates use innate immunity during theinitial phases of stress response because it takes several days toactivate specific immunity that provides threat specific antibodies andlymphoid cells immune regulation is mediated through numerous signalingproteins called cytokines or interleukins. Increased immunity can bebeneficial (e.g., short-term immune activation that removes a bacterialinfection) or harmful (chronic inflammation and autoimmunity increasephysiological stress through oxidative stress and apoptosis).

Microbial Activation (10). This pathway monitors the activation ofstress responses in microorganisms (bacteria, fungi, viruses), andsignaling between microorganisms and host cells. Commensal microbialbiofilms are an integral part of animal and plant bodies and contributeto physiological homeostasis. In animals, microbial biofilms areprimarily associated with the inner and the outer body surthees (themucosal epithelium and the skin). Therefore microbial biofilms aresensitive both to environmental stressors (e.g., uv light) as well as tomicro-environmental conditions in host tissues and body fluids (e.g.,oxidative stress). During physiological stress, increased signalingbetween microbial biofilms and host cells promotes protection of theorganism through modulating host's stress responses. For example,signaling by gastrointestinal microflora modulates levels of proteinswith key roles in redox control, cellular detoxification, chaperoning,cell growth, apoptosis and immunity such as metallothionein, Hsp25,ferritin, p53, TGF beta, IL-8 and IL-10. When pathogenic microorganismsinvade animals or plants, their stress responses are elevated, which inturn increases stress responses in the host((bacterial heat shockproteins are animal superantigens). Disease-causing microorganisms alsorelease soluble mediators that trigger cellular stress and activatemultiple stress response pathways in infected as well as remote hosttissues (e.g., HIV Tat protein).

Osmotic Stress Response (10: This pathway involves the organism'sresponse to sudden change in the solute concentration around a cell,causing a rapid change in the inoveinent of water across its cellmembrane. Under conditions of high concentrations of either salts,substrates or any solute in the supernatant, water is drawn out of thecells through osmosis. This also inhibits the transport of substratesand cofactors into the cell thus “shocking” the cell. Alternatively, atlow concentrations of solutes, water enters the cell in large amounts,causing it to swell and either burst or undergo apoptosis

Stress Response (SR) Biomarkers

Activation of SR pathways by stressors results in a pattern of expressedmolecules such as genes, proteins, metabolites and lipids, referred toherein as “SR biomarkers. Accordingly, each of these biomarkers is saidto be “associated with” one or more SR pathways. Measuring the levels ofthese SR biomarkers provides useful information about the biologicaleffects of stressors. Preferably, the SR biomarkers are expressedmolecules such as proteins or fragments thereof, so long as the fragmentis capable of being recognized in an SR biomarker assay with the samesensitivity as the entire protein.

Preferred SR biomarkers and their known associations with SR pathwaysare listed in Table 2 and FIGS. 16 and 17A-17I. Additional SR Biomarkersand some but not all of their known associations with SR pathways arelisted in Table 3 and FIGS. 18A-18B.

TABLE 2 SR Biomarkers Association with SR Pathways and Expression inTaxonomic Groups of Organisms Abbre- viated Expression SR. Pathways # SRBiomarker Name A B C D E 1 2 3 4 5 6 7 8 9 10  1 Beta-endorphinEndorphin + + + 0 0 0 0 0 1 0 1 1 0  2 Caspase8 Caspase 8 + + + 0 0 0 10 0 1 0 1 0  3 Cyclin D1 Cyclin + + + 0 0 0 1 0 1 1 0 0 0  4Cyclooxygenase 2 Cox-2 + + 1 0 0 0 0 1 1 1 1 0  5 Cytochrome P 450CYP450 + + + + + 1 1 0 0 0 1 1 0 0 0  6 Cytoplasmic cytochrome cCytc + + 0 0 0 1 0 1 1 0 1 0  7 Epidermal growth factor receptorEGFR + + + 1 0 0 0 1 1 1 1 1 0  8 Ferritin Ferritin + + + + + 1 0 0 0 01 1 1 1 1  9 Glucocorticoid receptor GR + 1 1 0 0 0 1 1 1 1 0 10 Glucoseregulated protein Grp58 Grp58 + + 1 1 1 0 0 1 0 0 0 0 11 Glucoseregulated protein Grp75 Grp75 + + + + + 1 0 1 0 0 1 0 1 1 0 12Glutathione-S-transferase p GST + + + + + 1 1 0 1 0 1 1 0 1 0 13 Heatshock protein 25/27 Hsp25/27 + + + + + 1 1 1 0 0 1 1 1 1 1 14 Heat shockprotein 40 Hsp40 + + + + + 0 0 1 0 0 1 0 1 1 0 15 Heat shock protein 60Hsp60 + + + + + 0 1 1 0 0 1 0 1 1 0 16 Heat shock protein 90Hsp90 + + + + + 1 1 1 1 1 1 1 1 1 0 17 Heat shock transcription factorHSF-1 HSF-1 + + + + + 1 1 1 0 0 1 1 0 1 0 18 Heme oxygenase-1 HO-1 + + 10 0 0 0 1 1 1 1 0 19 Interleukin IL-1beta IL-1 + + 0 0 0 0 1 1 0 1 1 020 Interleukin IL-6 IL-6 + + + 1 0 1 0 0 1 0 0 1 0 21 Interleukin IL-8IL-8 + + 0 0 0 0 1 1 0 1 1 1 22 Interleukin IL-10 IL-10 + 0 0 0 0 1 1 01 1 1 23 Interleukin IL-12 IL-12 + 0 0 0 0 1 1 0 1 1 0 24 LamininLaminin + + 1 1 0 0 1 1 1 1 1 0 25 Leptin receptor ObR + 0 0 0 0 0 1 1 11 0 26 Metallothionein MT + + + + + 1 0 0 1 0 1 1 1 1 27Stress-activated MAP kinase Mekk-1 Mekk-1 + + + + + 0 0 0 0 1 1 0 1 1 28Mitogen activated MAP kinase Mek-1 Mek-1 + + + + + 0 0 0 1 1 1 1 1 0 29NADPH-cytochrome P 450 reductase CYP red + + + + 1 1 0 0 1 1 1 1 0 30Nitric oxide synthase II, inducible iNOS + + + + 1 0 0 0 0 1 1 1 1 1 31Proto-oncogene c-Fos protein Fos + + + 0 0 0 in 1 1 1 1 1 0 32Proto-oncogene c-Jun protein Jun + + + + 0 0 0 in 0 1 1 1 1 0 33Serotonin receptor 5HT R + + + + 1 0 0 0 0 1 1 1 1 0 34 Serotonin5HT + + + + 0 0 0 0 1 1 0 1 1 0 35 Substance P Substance P + + 0 0 0 0 11 0 1 1 0 36 Superoxide dismutase Mn SOD Mn + + + + + 1 0 0 1 1 1 0 1 11 37 Superoxide dismutase CuZn SOD Cu/Zn + + + + + 1 0 0 1 1 1 0 1 1 138 Transforming growth factors beta-1,2,3 TGF + + + + 0 0 0 0 0 1 1 0 11 39 Tumor suppressor p53 p53 + + + 0 0 0 1 0 1 1 0 0 1 40 Vasoactiveintestinal peptide VIP + + 0 0 0 0 0 1 0 1 1 0

TABLE 3 SR Biomarkers: Association with SR Pathways Abbreviated SRPathways # SR Biomarker Name 1 2 3 4 5 6 7 8 9 10 41 Heat shock protein70 Hsp 70 + + + + + 42 Matrix metalloproteinase 9 MMP + + + 43 Aldosereductase ALR + + + + 44 Apoptosis signal-regulating kinase 1 ASK + 45Aquaporin 5 AQP + + + 46 Betaine GABA transporter 1 BGT + + + + + 47SAPK SAPK + + 48 Caspase recruitment domain protein 9 CARD + 49 P38 MAPKp38 + + 50 Peripheral benzodiazepine receptor PBR + + + + + 51 Salivaryalpha-amylase SAA + 52 GroEL GroEL + + 53 Superoxide dismutase FC SODEC + + + 54 Cell adhesion molecules V-CAM, 1-CAM + + 55 Monocytechemotactic protein 1 MCP + + 56 Catalase Cat + 57 Hypoxia inducedfactor 1 alpha HIF-1 + 58 Glutathione peroxidase GSHPx + 59 Carbonicanhydrase CAA + + 60 Ornithine decarboxylase ORD + 61 Vasoendothelialgrowth factor VEGF + + 62 Erythropoietin EPO + + + 63 MelatoninMelatonin + + 64 Thyroid-stimulating hormone receptor TSHR + 65Methenyl-tetrahydro-folate reductase MTHFR + 66 Oxytocin Oxytocin + 67Thromboxane synthase 1 TBXAS1 + + + 68 C-reactive protein CRP + 69TNF-alpha TNF + 70 Apolipoproteins A and B apo + + + + 71 Toll-likereceptor TLR + + 72 TspO protein TspO + 73 Bacterial trehalose synthaseTre-6P + + 74 Bacterial Sigma S factor RpoS + 75 Protease DegP DegP + 76Superoxide dismutase Fe SOD Fe + + + 77 Glutathione reductase A gorA + +78 Ferric uptake regulator fur + + 79 Multidrug efflux pump aef + + 80Sigma-B factor Sigma B + 81 DNA-binding protein stationary phase dps +82 DnaJ DnaJ + + 83 GroES GroES + + 84 8-hydroxy-deoxyguanosine8-OH-dG + 85 8-hydroxy-guanine 8-OH-G + 86 DNA damage binding protein-2DDB2 + 87 Xeroderma pigmentosum (XP) C protein XPC + 88 DNA glycosylaseOGG1 OGG1 + 89 pyrimidine-base DNA glycosylases NEIL + 90 uracil DNAglycosylase UNG + 91 thymidine DNA glycosylase TDG + 92 DNA glycosylaseMTH1 + 93 Apurinic/Apyrimidinic endonuclease APE + 94 MSH-2 MSH-2 + + +95 MLH-1 MLH-1 + + + 96 Senescence-associated beta-galactosidaseSA-beta-gal + + + 97 P21 p21 + + +

The relationship between each individual stressor and the eleven SRpathways, and thus the SR biomarkers associated therewith, may notalways be known, especially since the effects of many stressors onparticular SR pathways is not yet well studied. For example, the effectsof bird flu virus, engineered nanoparticles, and effects of deep spaceand deep sea or other extreme environments on each individual SR pathwaymay not be completely elucidated.

However, most SR biomarkers associated with the 11 SR pathways areuseful targets in assays to analyze the effects of both known andunknown stressors, such as environmental stressors and/ordiseases-related stressors. Accordingly, SR biomarkers associated withSR pathways are suitable targets for studying the effects of unknownstressors because they provide a response-oriented detection strategythat does not require prior knowledge of the stressor.

SR Biomarkers associated with the SR pathways are also suitable targetsin studying the of cts of complex stressors, some of which may be knownand others of which may be unknown. These complex, or “combined”stressors, are common in real-life scenarios, and may include multipleknown and unknown adverse conditions. Global warming, ozone holes, humaneffects on wildlife, urban pollution, natural and industrial disasters,poverty and war are examples of complex, combined stressors.

-   -   2. Dehydration

Dehydration is a water and electrolyte disorder that can severely affecthuman performance and health¹⁸⁻²¹. Preventable dehydration affects over90 million people in the US and the costs exceed 10 billion annually forunnecessary hospitalizations and avoidable complications¹⁸⁻⁵⁵.Dehydration includes hypertonic dehydration, isotonic dehydration andhypotonic dehydration. 3% dehydration (i.e., the loss a3% total bodywater) is a critical end-point for dehydration diagnostics in fieldsettings because it has measurable negative health consequences but itcan be simply treated by oral rehydration^(19,21,26,38,56-57).3%dehydration can be caused by sweating during strenuous physical workwith restricted fluid intake or by extremes of temperature, humidity oraltitude^(19,21,29-30,58). This type of dehydration frequently affectssoldiers, athletes, construction workers, policemen andfirefighters^(21,24,26,28,30,44,53,59-63). Acute dehydration due togastroenteritis is frequent in children^(34,36,39-42,46). Chronicdehydration associated with oral disease is a common side-effect ofcancer therapy or diuretics in diabetic and dialysispatients^(31-33,45). Elderly are at a greater risk for dehydrationbecause the mechanism controlling thirst becomes less sensitive withage, and dehydration occurs more rapidly due to a lower water content inthe aging body^(25,47,49,51,64). Many patients in the terminal phase oftheir illness experience dehydration due to a variety of causes relatedto their disease or treatment^(32-33,48). Dehydration is a major causeof mental deterioration and death in patients with Alzheimer'sdisease^(35,64). Life-threatening complications of dehydration includeheat stroke, heat illness and hyponatremia due to over-aggressiverehydration^(19-21,27-28,30,44,53,61,65). Dehydration is treated byrehydrating the subject, i.e., by drinking fluids or i.v. salineadministration.

Dehydration and hyponatremia are common in patients with cystic librisis, kidney, heart and liver disease. Currently used methods fordetecting dehydration are based on laboratory analysis of blood, urine,saliva and anthropometric indices such as body mass measurement. Thegold standards for dehydration assessment are blood osmolality, bodymass loss and TBW measurement using isotope dilution. However, nodehydration test is currently available for wellness and diseasemanagement in POC settings and in field conditions. A field-expedientdehydration test is also needed for monitoring the performance andhealth of military service members during training and deployment. Thus,there is an unmet need for non-invasive, rapid and accurate test for ≥3%dehydration that could be administered frequently to monitor thehydration status of at-risk individuals in fieldsettings^(18-19,24,44,59).

The dehydration test provided herein based on salivary SR biomarkersrisk is radically different from current dehydration tests because itutilizes a new assay principle based on based on monitoring thephysiological status of the patient. Recent studies indicate that thehomeostatic processes monitored by SRP are also activated bydehydration. As shown in Table 4, SRP biomarkers are relevant to thesensing of water loss. The assay principle was reduced to practice usingan immunoassay of SR biomarkers that monitor the physiological statusbased on cellular stress responses in saliva. The saliva test isnoninvasive, rapid and inexpensive, in contrast, the current dehydrationtests monitor physical properties (e,gs, osmolality) using blood orurine samples and expensive, time consuming laboratory assays. Asdescribed below in the Examples, selected dehydration markers includeAcidic Trehalase-like protein 1 (ATM), Osmotic stress protein 94 (OSP94)and Sodiuminiyo-inositol cotransporter (SMIT).

TABLE 4 SRP Biomarkers and Molecular Sensing of Dehydration Cellular andMolecular Effects of Dehydration Homeostatic Process SRP Efflux ofintracellular water Chaperoning + Increased intracellular salinity Redoxcontrol + Cell membrane distortion Cell adhesion and motility +Macromolecular crowding and denaturation Chaperoning + Increasedxenobiotics production Cellular detoxification + Oxidative stress Redoxcontrol + Cell growth arrest Cell growth + Pro-apoptotic signalingApoptosis + Hormonal changes Neuro-endocrine signaling + InflammationImmunity + Microbial biofilm changes Microbial activation +

-   -   3. Occupational Stress

Some occupations involve exposures to complex environmental andpsychological stressors, for example astronauts, pilots, divers,soldiers, police and haz-mat personnel. These occupational stressors aretypically much more diverse and higher, compared to mainstreamprofessions. Occupational stress can have adverse effects on health andperformance and therefore needs to be monitored. Currently, there are notests for occupational stress.

Occupational stressors can be physical (radiation, health, cold,altitude, gravity, vibrations), chemical (low air oxygen, toxicchemicals, tnicronutrient deficiency), biological (pathogens, injuries,jet leg, sleep deprivation) or psychological. Often, they involveundefined factors (e.g., outer space radiation, new pathogens),

There are two basic strategies for the detection of occupationalstressors. The first one measures levels of potential health threats inthe environment (e.g., levels of toxic chemicals). This approach is notsuitable for synergistic stressors (e.g., a mixture of individually safechemicals can be toxic), undefined agents (e.g, new types of pathogensor space radiation) and psychological stressors. The second strategy isbased on measuring changes in health status during and after exposures.Currently used health monitors measure vital signs and perform metabolicblood/urine tests. These monitors are not sensitive to many occupationalstressors and often they are invasive and not practical (e.g., a nest ofwires for vital signs). New health tests under development ine.asuremolecular indicators of immunological status (e.g., cytokines and latentviruses in saliva and blood) or assess cognitive pertbrmance (e.g.,specialized computer games).

The device of the invention offers a better solution for the assessmentof complex occupational stressors than any of the current methods. Themain advantages of SRP are broad-based sensitivity and new insights intothe mechanism of stress. The SRP sensitivity allows an upstream, earlydetection strategy for a broad spectrum of occupational stressors, incontrast, current methods provide a downstream detection strategyfocused on delayed effects. For example, SRP can measure moleculareffects directly triggered by a radiation exposure such as increasedfree radical levels and protein denaturation. These effects precedeimmunological or cognitive changes by several hours to several days. Theinsights into the mechanism of occupational stress could be used for thedevelopment of countermeasures,

-   -   4. Male Fertility Test

Current male fertility tests use sperm counts and blood and salivaassays for reproductive hon:nones. Semen is a complex mucosal fluid withmultiple cell types similar to saliva and milk. The device of theinvention can be used to measure cellular stress in semen as anindicator of sperm health and a predictor of rmde fertility.

-   -   5. Embryonic Health Test

Current prenatal health tests use amniotic cultures to perform FISHassays genetic abnormalities. SRP-based cellular stress test of theamniotic culture could serve as a new indicator of embryonic health anda predictor of prenatal health.

-   -   6. Oral Health Test

Current oral health tests use X rays and dental exams to detectperiodontal disease, a serious chronic health problem. New expensivegenetics tests are under development. SRP-based cellular stress insaliva could serve as a new indicator of oral health and a predictor ofperiodontal disease.

-   -   7. Saliva as a Diagnostics Sample

Oral diagnostics is a rapidly growing field that provides a convenientalternative to blood sampling for a rapidly expanding list of analytesand diseases, including an early test for a heart attack, it haspreviously been reported that SRP biomarkers in saliva were sensitive tochronic diseases and post-traumatic psychological stress. Saliva can besampled simply and noninvasively. A typical saliva sample (10 drops,about 0.3 ml) contains about a half million of epithelial and whiteblood cells. Salivary cells may play a role in the cellular andmolecular mechanism of oral disease transmission. It was also found thatsalivary cells express SRP biomarkers and that salivary SRP levels arestrongly increased by stress.

Saliva diagnostics provides a noninvasive and safe alternative to bloodor urine diagnostics¹⁻³. The main challenge of using saliva as adiagnostic sample is the inherently low concentration of solublebiomarkers in the cell-free saliva fluid¹⁻³, which is the currently usedmethod for saliva sampling¹⁻³. Whole saliva comprises all normalsalivary cells types including epithelial cells, monocytes, B and Tlymphocytes and granulocytes, We tound that whole saliva contains alarge number of cells (about 10⁶/ml) that contain. clinicallysignificant biomarkers⁴⁻⁷. Therefore, whole saliva samples that containsalivary cells provide a radically improved diagnostic sample for salivadiagnostics compared to samples which do not contain whole saliva cells.

Saliva samples may be collected by the subject or may be collected by apassive method in which a health care worker or person other than thesubject collects the sample. For example, unstiniulated saliva samplesmaybe be collected having the subject collect saliva into a sterilecontainer. Alternatively, saliva samples may be collected using a devicesuch as a small oral brush by brushing teeth and gum surfaces on bothsides of the mouth for about 20 seconds. To collect a larger volume ofsaliva, the brushing may be repeated using additional brushes. Acomparison of saliva samples collected using self-brushing, and brushingperformed by an assistant, showed that both methods yielded the sameaverage sample volume and cellular composition. The passive salivasampling method (brushing performed by an assistant) enables salivadiagnostics in subjects that cannot actively participate in activesaliva sampling such as infants, elderly, unconscious patients ormentally ill patients. Currently, no other passive saliva collectionmethods were published that enable saliva diagnostics in these subjects.

The device thr collecting salivary cells may consist of a disposablebrush and a reusable handle. The brush is suitable tbr oral use. Thebrush may be used to collect saliva by brushing gums and teeth, and thenmay be used to process the saliva into smears on slides or lysates.

Methods that collect saliva samples containing salivary cells provide aradical improved diagnostic sample for saliva diagnostics becausesalivary cells contain clinically significant biomarkers (e.g., proteinsand DNA)⁴⁻⁷. In contrast, current methods for saliva sampling collectcell-free saliva that has inherently low concentration of solublebiomarkers, which is the main challenge in using saliva as a diagnosticsample¹⁻³.

In one embodiment of the invention, there are provided referencereagents and materials for saliva diagnostics generated by inducingcellular stress in cultured normal salivary cells by in vitro treatment.Although as described below, the induction of proteomic biomarkers using5 stressors is exemplified, the treatment principles could easily beadapted to protocols that use different stressors, or other stressmarkers such as MRNA, DNA, reporters or small molecules associated withthe activation of SR pathways (see Table 1). Moreover, the methodprinciples could be also easily adapted for other human cells that havea diagnostic value, white blood cells, and thr diagnostic cells fromanimals, plants and microorganisms. Although the production of cellsmears as reference materials is exemplified, the method principlescould easily be adapted to the production of other types of referencematerials and reagents such as protein lysates, mRNA lysates orcell-free saliva fluid.

Methods that induce cellular stress in cultured normal salivary cells byin viim treatment provided novel reference reagents materials tbr salivadiagnostics such as salivary cell smears with normal and increasedlevels of salivary biomarkers. The in vitro production of referencereagents and materials was rapid, convenient and inexpensive. The newreference materials are radically different from current referencematerials produced using cell-free saliva samples¹⁻³ collected frompatients with and without specific medical conditions.

Also provided herein are methods for the development of salivarybiomarker assays. In one example, the method uses reference reagents andmaterials prepared as described in Example 2. Two types of laboratorysaliva immunoassays are exemplified, the immunocytochemical (ICC) assayand the ELISA assay. Together, these assays enable the development andvalidation of saliva biomarker panels because they provide complementaryanalysis of cell-associated (ICC) and soluble (ELISA) saliva biomarkersas described in Example 5. In addition, laboratory saliva ELISA assaysare also useful as reference assays for the development of commercialsaliva assays such as the lateral-flow immunoassay (LFIA) test strip.Although as described below, the development of saliva. ICC and ELISAassays exemplified, the principles of the method could easily be adaptedto developing other types of immunoassays such as LFIA, and to assaysthat measure other types of markers such as mRNA, DNA or smallmolecules. The method for developing new saliva assays using referencereagents and materials produced in vitro was rapid, convenient andinexpensive compared to currently used methods based on clinical salivasamples. Saliva assays produced by the new method use saliva samplesthat contain salivary cells and therefore the new assays are radicallydifferent from current saliva assays based on cell-free salivasamples¹⁻³.

Also provided are methods for measuring, baseline concentrations ofsaliva biomarkers using two complementary assays, ICC forcell-associated biomarkers rn saliva smears, and ELISA for solublebiomarkers in saliva lysates. In one example, the method uses referencereagents and materials prepared in vitro as described in Example 2.Biomarker baselines in normal saliva provide a useful benchmark theconstruction of biomarker panels for saliva diagnostics as described. InExample 5. Although as described below, the baseline measurement of aprotein biomarker using the ICC and ELISA assays is exemplified, theassay principles could easily be adapted to measure soluble proteinmarkers using other assays such as the lateral-flow immunoassay, andother types of biomarkers such as mRNA, DNA or small molecules.

Also provided are methods for constructing a biomarker panel that isuseful for salivary diagnostics of health disorders. Although asdescribed below, panels of proteomic markers are exemplified, the sameprinciple could easily be adapted to measure, for example, mRNA, DNA orsmall molecules, and to assess the diagnostic value of the salivabiomarker panel using other statistical methods than exemplified here.The new method is rapid, convenient and inexpensive because it usesreference reagents produced in vitro, compared to current methods thatuse reference reagents based on clinical saliva samples¹⁻³.

-   -   8. LFIA Biosensors

The firstFDA-approved commercial oral biosensor broke the ground for asuccessful commercialization of oral diagnostics. This breakthrough testis ORAQUICK ADVANCE® RAPID HIV-1/2 ANTIBODY TEST produced by OraSure.The test is based on a mature biosensor technology, the lateral-flowimmunoassay (LFIA). LFIA is rapidly expanding to provide a wide varietyof POC and point-of-need tests for wellness (pregnancy and ovulationtests), public health (HIV and Hepatitis C tests) and tbrensics (drugand alcohol tests).

The present invention provides a simple analyzer that provides aquantitative measurement of physiological stress. Examples of theanalyzer is shown in FIGS. 6 and 7A-7B. The analyzer consists of areusable reader with a digital result display and disposable cartridges(test strips). Alternatively, the whole unit is disposable. The testresult is a “stress number,” or “SRP score,” which is a quantitativeindicator of the general stress level. A color-coded results window isused for simplified interpretation of the stress number: e.g., green fornormal, yellow for mild/moderate red for high stress levels (similar toa digital thermometer). If the stress number is above the normal range,a digital message prompts the user to obtain advanced analysis of theirstress, and to seek medical advice.

In one embodiment of the analyzer device, there is provided a pen-sizedigital device consisting of a disposable test strip, a reusableelectronic reader and a clip-on holster. The user briefly puts the endof the test strip in the mouth to collect saliva and then inserts thetest strip into the reader. In less than 3 minutes, the reader shows adigital stress measurement. A color guide shows whether results indicatenormal health (green), a mild health problem (yellow) or serious healthdeterioration (red). Results from multiple tests are stored on board andcan be wirelessly communicated to a remote care center. The deviceoperates autonomously using small batteries that last for several weeks.

An advanced analyzer that provides quantitative and qualitativemeasurements of physiological stress is also provided in the presentinvention. The analyzer uses a disposable cartridge to perform a highlymultiplexed immunoassay (10-40 individual biomarkers). Assay results aremeasured and processed using an opto-electronic reader, Test results aredisplayed digitally. The analyzer is equipped for data transfer (USB,Bluetooth) to a web-based service (Stress Net) that supports advancedanalysis of test results. The analyzer can be a desktop device or aPDA-class handheld device.

The average SRP score is computed by the device based on measurements ofthe individual biomark.ers and displayed in the results window at theend of the test. The average SRP score will be interpreted as aquantitative indicator of the general stress level, analogous to the“stress number” (combined. SR blomarker score), since the average SRPscore strongly correlated with combined SR biomarker scores in referencesamples. If the general stress level is above the normal range, the userwill be prompted to pertorm advanced analysis of his SRP profile using aweb service. The software on the web service interprets the SRP profileand provides user-friendly results in the form of a short message. Themessage (1) describes the nature of personal stress, e.g., the % risk ofspecific health problem, (2) shows top three personal stress drivers,(3) recommends wellness productsisewices that are the best match for thepersonal stress drivers, and (4) directs the user to additionalinformation,

-   -   9. Predictive Medical Diagnostics and Personalized Disease        Management

Many health problems and diseases do not have a lab test that couldprovide early warnings before the onset of clinical symptoms. This isparticularly true for mental diseases. Often, the earlier a disease isdiagnosed, the more likely it is that it can be cured or successfullymanaged. Managing a disease, especially early in its course, may lowerits impact on life or prevent or delay serious complications. Diseasemanagement strategy strongly depends on the ability to predict theseverity of a disease, for example, differentiating metastatic cancer,Alzheimer's dementia, or kidney failure. Such predictive tests arecurrently unavailable.

Chronic diseases are associated with early physiological changes thatmight be detectable using SRP bioniarkers in saliva. Early disease testscan be done based on stress-induced changes in microflora and mobilegenetic elements (MGE). SRP profiles might reflect the course of adisease. For example, a metastatic cancer might have a different SRPprofile than a non-metastatic cancer because metastatic cells producechemical compounds and biological interactions that are likely to affectstress responses in the normal cells and biofluids analyzed in SRPassays.

Methods: The SRP panel (40 or 41 biomarkers) is analyzed in samples fromcase/control subjects (cases represent a disease, e.g., breast cancer,BC). If SRP profiles unambiguously discriminate between BC and controls,the panel is minimized toward the smallest panel sufficient for the. BCclassification. Based on the preliminary data, the i:ninimized panelwill consist of 3-6 biomarkers. If SRP profiles from the original. SRPpanel do not discriminate BC, new biontarkers are added to the paneluntil the SRP profile can discriminate BC. The new biomarkers areselected using the SR pathway profile of BC determined by the originalSRP panel. For example, if BC-related stress preferentially involvesmisfolded proteins, oxidative stress and changes in cell cycle andgrowth, then new biomarkers tbr these processes will be preferred. WhenSRP profile can discriminate BC, the biomarker panel will be i:ninimizedas described above. The minimized panel is detected using combined SRbiomarkers (“multi-SRP assay”), if a combined score is sufficient for BCdetection. Alternatively, the biomarkers in the minimized panel aremeasured individually , if a SRP profile is needed for BC detection. Amulti-SRP test or an assay for 3-6 individual SRP biomarkers will beimplemented using the device of the invention.

-   -   10. Human Disease Research

The cellular and molecular mechanism of a disease (i.e., molecularpathogenesis) shows where and how the disease harms the body. Diseasesperturb cellular and physiological homeostasis, and the perturbationpattern might be disease-specific and reproducible (i e., adisease-specific stress signature). Disease countermeasures prevent,reduce or remove the disease-specific stress signature. Disease-specificstress signatures can be analyzed by SRP. This information can be usedto guide the development of disease countermeasures: preventivetreatments, diagnostics tests and therapeutic interventions. Molecularpathogenesis of most diseases remains elusive including the Spanish fluand AIDS. New molecular and bioinformatics tools are needed, inparticular systems biology-based approaches such as SRP measurementsusing the device of the invention.

-   -   11. SRP Tests for Cancer: Oral Tests for Cancer Screening and        Lab Tests for Cancer Diagnostics

Carcinomas of the skin, breast and prostate are among the most commonhuman malignancies. Current diagnostic methods typically involve aseries of tests. For example, a general screening test at POC is orderedby a family doctor during routine health testing (e.g., mammogram forBC). If this test is positive, the patient is referred to a CancerCenter. The Cancer Center surgeon performs a surgical biopsy that isexamined by a cancer pathologist using morphological criteria nuclearmorphology, mitotic number) and molecular pathology (antibody stainingor DNA probing). If the biopsy is considered positive (malignantgrowth), the patient is referred for additional surgery. The surgicalspecimen is analyzed by a pathologist to assess the tumor grade based ongeneral grading scales (e.g., Gleason score for PC). New research inpersonalized medicine aims to delineate the molecular mechanism ofindividual tumors using gene arrays or antibody staining. Followingradio- and chemotherapy, diagnostic tests based on surgical biopsies andblood assays (not available tbr most cancers; PSA is used for PC) andare used to identify metastatic cancer. Many cancer survivors sufferchronic pain and mental health problems for which there are no objectivetests. A related health problem is care giver stress.

Tests for SRP are applicable across the cancer managementcontinuum—examples are listed below.

(1) Cancer Screening tib Noninvasive Early Detection of Cancer,

-   -   Rapid oral test for BC and PC: a disposable LFIA. The test is        based on a multi-SRP assay optimized for a particular cancer (a        yestno result). Some versions can discriminate metastatic        cancer.    -   Rapid semen test for PC: a disposable LFIA. The test is based on        a multi-SRP assay optimized for PC (a yes/no result). Some        versions can discriminate metastatic cancer.    -   Lab test for cervical cancer. The test is based on a multi-SRP        staining kit optimized for cervical cancer detection in a        standard cervical smear. Some versions can discriminate        metastatic cancer. The kit is used to stain a duplicate cervical        smear in parallel with the standard PAP test and provide a        yes/no result. The SRP test could be used as a decision support        for PAP (increased accuracy of cancer prediction) and ultimately        replace the PAP test. The accuracy of PAP would be improved in        two ways. First, positively staining cells with abnormal        morphology (i.e., PAP test reading made easier, faster, i:nore        accurate). Second, positively staining pre-cancerous cells with        a normal morphology that cannot be detected by PAP. The        replacement could happen fast if the SRP test would provide an        outstanding benefit over. PAP, e.g., discriminating metastatic        cancer.

(2) Cancer Diagnosis for Early Detection of Metastasis.

-   -   Lab test for a particular cancer. The test is based on a        multi-SRP staining kit optimized for the particular cancer and        corresponding tissue type. The kit is used to stain the biopsy        tissue and also the surgically removed tissue (tumor and        adjacent tissue). The test can discriminate metastatic cancer.    -   A staining kit for measuring individual SRP biomarkers. Results        (SRP profi es) interpreted using the computer software. Results        show a match with reference SRP profiles (i.e., discriminate        between not cancer, cancer, metastatic cancer). The results        indicate the molecular mechanisms of the particular cancer        (pathway profile). This information can be useful as a guide for        personalized.    -   (3) Cancer Survivors for improved Quality of Life.        -   Rapid oral test for cancer-related psycho-biological stress            in survivors.        -   Rapid oral test for cancer care giver stress.    -   12. Early PTSD Test

A large number of military service members suffer from PTSD andtraumatic brain injury (TBI). These diseases are complexpsycho-biological disorders that are hard to detect and quantify,particularly in early stages, before they can be assessed using standardpsychological neurological tests. These standard tests require highlytrained medical personnel, therefore they are not practical for POC orat home monitoring. New tests for PTSD (under commercial development)include assays for saliva cortisol and saliva alpha-amylase (SAA). Theseanalytes are biotnarkers for HPA and sympathetic nervous systempathways. SRP covers these pathways and many more pathways that arelikely to be activated by psycho-biological stress. Cortisol and SAA arealso likely to give false positive signals because they are increased byroutine stressors.

-   -   13. Early Alzheimer's Test

Alzheimer's disease is the most common cause of dementia. The device ofthe invention can be used for rapid oral test for early Alzheimer's. Thetest is based on SRP biomarkers optimized for the disease.

-   -   14. Early Autism Test

Autism (ASD) is a rapidly growing health l roblem for kids in the US.Current goal for autism diagnostics is a test suitable for infant (<3yrs). Oral SRP test using the device of the invention would be moresuitable than tests currently under development.

-   -   15. Early Kidney Stones Test

A July 08 report predicted a sharp rise in kidney stones-related healthproblems in the US due to a higher incidence of dehydration caused byglobal warming. The device of the invention can be used for a rapid oraltest for early detection of kidney stones. The test is based all SRPbiomarkers optimized for kidney stones.

-   -   16. Early Kidney Disease Test

Chronic kidney diseases (CU)) are on a rise worldwide and earlydetection is essential for CKD management. The device of the inventioncan be used for a rapid oral test forr early detection of CKD. The testis based on SRP biomarkers optimized for CKD.

-   -   17. Animal Wellness and Food Safety

There is a worldwide increase in emerging diseases and environmentalstressors related, to factory farming, genetic modifications of cropsand life stock, antibiotics overuse, human impact on wildlife and globalwarming. This increase might be responsible for increased morbidity andmortality of domesticated and wild animals, wild species extinctions,and collapses of the US lobster fishery and the honeybee industry. Earlydetection of new diseases and environmental stressors is essential forpublic health, agricultural safety and wildlife protection andmanagement.

Factory farms keep animals in unhealthy conditions. Many animals arediseased and exposed to environmental stressors (crowding, injuries,imtibiotics etc.). Organic farms are more likely to have less stressedanimals. Besides the ethical objections, products from stressed animalsare likely to have a lower nutrient value and inight even present ahealth threat for consumers because stressed animals are likely to beill and the product might transmit diseases (e.g., mad cow disease), ormight contain metabolites that can cause health disorders because theyderegulate human cell functio For example, human metabolic disorderstriggered by animal hormones or immune disorders triggered by animalstress proteins. Currently, there is no objective test that regulatorsor consumers can use to assess whether a farm product is from a healthyor from a stressed animal. Such test would help consumers to chooseproducts from minimally stressed animals, and help regUlators to searchfor unhealthy farm conditions.

A related issue is the safety of animal foods (pet food, farm feed). Ifanimal foods contain parts of diseased animals, it might transmit thedisease (e.g., BSE) or cause other types of health disorders.

The device of the invention can be used for SRP analysis of animal cellsand tissues related to disease, injury, environmental stress, and animalhealth and product safety, including:

-   -   1. Stress Tests for Animals and Animal Products (Milk, Egg).        -   The tests use: the device of the invention modified for the            use with animal samples.        -   Saliva test for companion animals (dog, cat, horse); one            smart test device with species-specific cartridges or            sample-specific tests.        -   Milk test: a health test for dairy animals & milk quality            test. Could be made as an attachment for a milking machine            or equipment for milk quality test. SRP assay of milk cells            and fluids similar to the saliva SRP assay.        -   Egg test: a health test for poultry & egg quality test.        -   Male fertility test.        -   Urine test (a litter test fir cats; farm animals).        -   Fish test. Fish biopsy or a surrogate small fish or            invertebratelying in the same habitat.        -   Shellfish test.        -   Beehive wellness test. The honeybee keeping industry has            nearly collapsed due to an unknown health threat            (environmental factors or disease).    -   2. Stress Test for Meat.        -   Cattle, pork, poultry, fish.    -   3. Stress Test for Animal Food.        -   Multi-SRP test that shows whether pet food and farm feed            products contain parts of stressed animals.        -   4. Stress Test for Sentinel Organisms,        -   Multi-SRP test of sentinel animals and wild animals found            sick or dead of unknown causes. Increased stress indicates            exposure to an emerging/occult disease or environmental            stressors. For example, chickens are currently used as            sentinels for infectious bird diseases.    -   18. Agricultural Safety

Plant diseases have increased worldwide possibly due to unsustainablefarming practices such as the widespread use of chemical fertilizers,pesticides and genetically modified plants, and global warming.Integrated monitoring of soillplanticrop health could improve wellnessof farm crops, garden plants and house plants, and provide decisionsupport for the protection and management of wild plant ecosystems suchas forests and wetlands. Healthy crop plants are likely to produce cropsthat are more nutritious and case less health problems such as foodallergies. Currently, there is no objective test that regulators orconsumers can use to assess whether a crop (grains, fruits etc) is froma healthy or from a stressed plant. Such test would help consumers tochoose products from minimally stressed plants, and help regulators tosearch for unhealthy crop conditions.

Soil fertility correlates with the microbial richness of the soil, whichis naturally low in some soils (e.g., tropical soil) and becomesdepleted by agricultural use. Currently, there is a strong globalinterest in the restoration of soil fertility due to two factors arapidly growing need for increased food production, and soil loss &depletion driven by global warming. However, there is no direct test formonitoring the wellness of soil microflora (the indirect test is theassessment of soil fertility based on crop yield and quality). There aretraditional soil probiotics such as compost or charcoal but no new,scientifically-based products that would rationally reduce stress insoil microbial systems.

There is an urgent need for a new technology that could assess andimprove soil fertility and crop health. The device of the invention toassess SRP technology meets these specifications.

-   -   19. Plant Disease and Food Research

Multi-SRP and SRP assays using the device of the invention can beadapted for soil and plant samples.

-   -   20. Stress Tests for Soil, Plants and Crops

Different soil samples can be analyzed using the device of the inventionwith the SRP biomarker panel optimized for soil microorganisms. AnotherSRP panel can be optimized for plants, focusing on stress markers forplant organelles (chloroplasts, mitochondria), commensal microorganisms(same as soil) plus stress markers that are in all species (e.g.,Hsp60:/GroES or SOD). Candidate samples for plants include: roots,leaves, sterns. sap. Samples for crops include: grains, fruits.

-   -   21. Environmental Safety

Water Safety

Water safety affects public health, aquaculture, agriculture and naturalaquatic ecosystems. Standard methods for assessing water safety arebased on measuring several chemical and physical parameters (e.g., pH,temperature and turbidity), and levels of specific microbial andchemical contaminants. In England, a traditional method for water safetyis still used: the health condition of fish in the water (how many, howfast they swim past the observatory). Frogs are also traditionally usedas sentinel organisms tier freshwater health. Since these methods do notprovide early warnings of declining water safety, new methods arecurrently being developed and tested. Several new methods measure thehealth condition of native aquatic microorganisms. One method uses aninfrared motility monitor. Another one, Aqua Sentinel, developed at theOak Ridge National Labs, uses a fluorescence reader to measure changesin algal bioluminescence.

The device of the invention can be used to provide SRP-based tests forwater safety, and provides several advantages such as: applicability toboth fresh water and seawater, broad-based sensitivity to changes inchemical, physical, biological water parameters including parametersthat are not measured by current sensors such as new types of airpollutants or agricultural run-off chemicals, early warning of incipientwater health deterioration (molecular stress responses precede changesin an algal fluorescent signature or motility), detection of emergingpathogens and bio toxins in aquaculture water that could be directlycorrelated to fish health, signature can be used to diagnose the natureof the water stress and to recommended countermeasures and used tomonitor the effectiveness of countermeasures in restoring water health.

Background—Air Safety

Microbial biosensors for airborne toxins have been introduced recently.Typically, these are genetically engineered bacteria or recombinantflies with a read-out gene (e.g., lux) linked to one of severalparticular toxin-sensitive genes (hsp70, DNA J). The disadvantage isthat recombinant organisms have to be manuflictured for the biosensoroperation, and the small set of recombinant sensor genes in thebiosensor might not be sensitive to the large and diverse spectrum ofenvironmental stressors that affect people. The device of the nventioncan be used to provide SRP-based tests for air safety,

-   -   22. Space Technology

Key areas in space biology research that are useful with the device ofthe invention include: diagnostic and therapeutic technologies forastronaut health. The goal is to identify health risks of space flightand develop countermeasures to reduce those risks. The device of theinvention can also be used for fundamental space biology investigationsin microbial, plant and cell biology and animal physiology, i.e., howlife responds to gravity and space environments. Additionally, thedevice of the iliVention can be used to detecting life's signatures forfuture planetary missions to Mars, Europa and Titan.

-   -   23. Detection of Life's Signatures

Stress responses (SR) are universally present in all organisms on Earth.Responses to universal stressors are essential for life in general andcould be used in the search for life. The universal stressors arephysical-chemical gradients or agents commonly present in planetarygeological environments (e.g., electromagnetic radiation, radioactivity,temperature, gravity, heavy metals, water, CO₂). Universal stressors arealso generated by general biological processes such asself-organization, electron transfer and metabolism (e.g., heat, entropyand free radicals). Different biological systems might use verydifferent biochemical structures for SR but the different biochemicalstructures are likely to have the saine or similar physical-chemicalfunctions. These general functions can be deduced through comparativestudy of biochemical structures that are commonly used fOr SR byterrestrial organisms. Components of the general physical-chemical SRfunctions are used as biomarkers for life.

For example, free oxygen and nitrogen radicals (RONS) are universalstressors produced by solar radiation as well as a by-product ofelectron transfer and metabolic reactions. RONS cause harm to all livingsystems through damage to macromolecular structures and Shills in redoxbalance. All terrestrial organisms have SR against RONS. Mostprokaryotes and eukaryotes use control RONS using superoxide dismutases(SOD). The physical-chemical function of SOD is carried out by the metalmoiety of SOD, which contains an antioxidant heavy metal (Fe, Mn, Cu,Zn). These and similar (Ni, Co) metals are candidate biomarkers forbiological anti-RONS responses.

Another universal stressor is water loss (desiccation). Most prokaryotesand eukaryotes have a SR for adapting to life without water(Anhydrobiosis). If there was or is life on Mars, it had to deal withperiodical desiccation as well. The physical-chemical functionsunderlying the anhydrobiosis SR are candidate markers tier lifedetection,

Bacterial and eukaryotic, cells use organic osmolytes to cope withanhydrobiosis (glutamate, proline, glycerol, sucrose, trehalose,sorbitol, myo-inositol and glycine betaine). The physical-chemicalprinciple of the osmolytic compounds is structure-making (cosmotropic)function: they organize the water structure (hydrogen bonding) which isessential for structural integrity of biological membranes andbiopolymers proteins). The known organic osmolytes listed above, andother cosmotropic compounds, are candidate biomarkers for biologicalresponse to osmotic stress and for anhydrobiosis.

-   -   24. Monitoring HIV/AIDS Risk and Treatment Outcome

HIV/AIDS is a priority public health condition⁶⁷. There are about 1.1million HIV infected people the U.S and 56,300 new HIV infectionsannually⁶⁷. Currently, two lab tests (CD4 count and viral load) are thegold standards for assessing AIDS risk and guiding cART⁶⁸⁻⁶⁹. CD4 counts350 (recently increased to 500) and 200 cells/mm³ are standardactionable thresholds for guiding cART⁶⁸. The CD4 test is expensive,invasive, time consuming, requires specialized equipment, highly trainedpersonnel and has to be repeated every 3-4 months. Thus, there is anunmet need for an affordable POC test for AIDS risk and guiding cART⁶⁹.HIV/AIDS is typically treated with non-nucleosidase reversetranscriptase inhibitors, nucleosidase reverse transcriptase inhibitors,protease inhibitors, fusion inhibitors, CCR5 antagonists, integrasestrand transfer inhibitors or a combination thereof.

In one embodiment of the invention there is provided a rapid saliva testfor HIV progression (unsuppressed HIV) and acute HIV that has beendeveloped using following steps: (1) A candidate panel of 52 SR markers(see Tables 2 and 3) was constructed using methods from Example 5. (2)The initial clinical validation of the panel used methods from Example 5and saliva samples from HIV/AIDS patients (n=100) with CD4 counts >500,200-500 and <200 cells/mm³. The AIDS risk test based on salivary SRbiomarkers is radically different from current tests for the conditionbecause it utilizes a new assay principle based on monitoring thephysiological status of the patient. The assay principle was reduced topractice using an immunoassay of SR biomarkers that monitor thephysiological status based on cellular stress responses in saliva. Thesaliva test is noninvasive, rapid and inexpensive. In contrast, currenttests for AIDS prognostication monitor the immunologic, virological orgenetic status of the patient using blood samples and expensivelaboratory assays^(68-69,72). The saliva test for AIDS prognosticationhas potential to provide significant benefits for public health andsubstantial healthcare savings in several ways: (1) AcceleratingHIV/AIDS care and slowing the spread of HIV: Two rapid oral tests couldbe administered during routine healthcare screening, an HIV diagnosticstest followed by the saliva test in order to inform patients about theirHIV status and AIDS risk in a single office visit so that they couldreceive care immediately. Pain-free, afthirdable oral testing is likelyto increase the number of HIV/AIDS patients connected to care and fewerpeople be exposed to HIV: 54-70% of new HIV infections in USA are causedby people who are not treated and engage in risky behaviors because theydo not know that they are HIV infected⁶⁷. (2) Moving AIDS monitoringfrom lab to point-of-care will greatly improve the delivery of clinicalcare and medications in resource-limited settings where the standardtests for AIDS risk are not affordable and costly cART drugs aredelivered inefficiently, without lab tests^(69,74). (3) Enablingpersonalized HIV medicine: frequent affordable testing of cART efficacywill facilitate designing and modifying cART for individual patients,which has potential to improve treatment outcomes and decrease clinicalcosts⁷². Current treatment for HIV/AIDS include Non-nucleoside reversetranscriptase inhibitors (NNRTIs); Nucleoside reverse transcriptaseinhibitors (NRTIS); Protease inhibitors (PIs); Fusion inhibitors; CCR5antagonists (CCR5s); Integrase strand transfer inhibitors (INSTIs) andcombinations thereof. As described in the Examples below, biomarkers forHIV progression (unsuppressed HIV) and Acute HIV include Bone MarrowStromal Cell Antigen 2 (BST2), Salivary Agglutinin gp340 (SAG) andascular endothelial growth factor C (VEGF-C).

-   -   25. Traumatic Brain Injury

Traumatic brain injury (TBI), also known as intracranial injury, occurswhen an external force traumatically injures the brain. TBI can beclassified based on severity, mechanism (closed or penetrating headinjury), or other features (e.g., occurring in a specific location orover a widespread area). Traumatic brain injury (TBI) includes mild TBI(mTBI, concussion), severe TBI (sTBI) and neurocognitive disorder (NCI))due to TBI (NCDT). Brain trauma can occur as a consequence of a focalimpact upon the head, by a sudden accelerationldeceleration within thecranium or by a complex combination of both movement and sudden impact.In addition to the damage caused at the moment of injury, brain traumacauses secondary injury, a variety of events that take place in theminutes and days following the injury. These processes, which includealterations in cerebral blood flow and the pressure within the skull,contribute substantially to the damage from the initial injury. TBI cancause a host of physical, cognitive, social, emotional, and behavioraleffects, and outcome can range from complete recovery to permanentdisability or death. Some of the current imaging techniques used fordiagnosis and treatment include computed tomography and MRIs. Dependingon the injury, treatment required may be minimal or may includeinterventions such as medications, emergency surgery or surgery yearslater. Physical therapy, speech therapy, recreation therapy,occupational therapy and vision therapy may be employed forrehbilitation. Mild traumatic brain injuries usually require notreatment other than rest and over-the-counter pain relievers to treat aheadache. Moderate to severe traumatic brain injuries focuses on makingsure the person has an adequate oxygen and blood supply, maintainingblood pressure, and preventing any further injury to the head or neck.Medications to limit secondary damage to the brain immediately after aninjury may include: Diuretics; Anti-seizure drugs; and Coma-inducingdrugs. As described in the Examples below, biomarkers for NCDT includeCytochrome P450 Reductase (CYPOR), Natriuretic peptide receptor A (NPR)and Oxytocin receptor (OTR).

-   -   26. Post-Traumatic Stress Disorder

Posttraumatic stress disorder (PTSD) is a mental disorder that candevelop after a person is exposed to a traumatic event, such as sexualassault, warthre, traffic collisions, or other threats on a person'slife(en.wikipedia.org/wiki/Posttraumatic_stress_disorder-cite_note-DSM5-2).Symptoms may include disturbing thoughts, feelings, or dreams related tothe events, mental or physical distress to trauma-related cues, attemptsto avoid. trauma-related cues, alterations in how a person thinks andfeels, and increased arousal. These symptoms last for more than a monthafter the event. Those with PTSD are at a higher risk of suicide.Diagnosis is based on the presence of specific symptoms following atraumatic event. The Main treatments for people with PTSD arecounselling and medication. Antidepressants of the selective serotoninreuptake inhibitor type are the first-line medications for PTSD andresult in benefit in about half of people. Other treatments for PTSDinchid.e tricyclic antidepressants, atypical antidepressants and moodstabilizers.

-   -   27. Heart Disease

Heart disease includes acute heart failure (AHF) with preserved ejectionfraction (HF-pEF), AHF with restricted election fraction (HF-rEF) andatrial fibrillation. Heart failure (HF), often referred to as congestiveheart thiture (CHF), occurs when the heart is unable to pumpsufficiently to maintain blood flow to meet the body's needs. Signs andsymptoms commonly include shortness of breath, excessive tiredness, andleg swelling. The shortness of breath is usually worse with exercise,while lying down, and may wake the person at flight. Common causes ofheart failure include coronary artery disease including a previousmyocardial infarction (heart attack), high blood pressure, atrialfibrillation, valvular heart disease, excess alcohol use, infection, andcardiomyopathy of an unknown cause. There are two main types of heartfailure: heart failure due to left ventricular dysfunction and heartfailure with normal ejection fraction depending on whether the abilityof the left ventricle to contract is affected, or the hearts ability torelax. The severity of disease is usually graded by the degree ofproblems with exercise. Heart disease is treated withAngiotensin-converting enzyme (ACE) inhibitors (examples includeenalapril (Vasotec), lisinopril (Zestril) and captopril (Capoten));Angiotensin II receptor blockers (examples include Cozar); Beta blockers(examples include carvedilol (Coreg), metoprolol (Lopressor) andbisoprolol (Zebeta)); Diuretics furosemide (examples include Lasix);Aldosterone antagonists (examples include spironolactone Aldactone) andeplerenone (Inspra)); inotropes and Digoxin (Lanoxin). As shown in theExamples below, biomarkers for heart disease include Cyclooxygenase-2,Epidermal growth factor receptor, Leptin and MAP kinase Mek-1.

-   -   28. Kidney Disease

Kidney disease, also known as nephropathy or renal disease, is damage toor disease of a kidney. Nephritis is inflammatory kidney disease.Nephrosis is noninflammatory.nephropathy. Kidney disease usually causeskidney failure to more or less degree, with the amount depending on thetype of disease. Acute kidney injury has often been called acute renalfailure, although nephrologists now often tend to call it acu kidneyinjury. As used herein the term kidney disease includeschronic kidneydisease (CKD) and acute kidney injury (AKI). Kidney disease may betreated by various methods including angiotensin-converting enzyme (ACE)inhibitors, such as captopril (Capoten), enalapril (Vasotec), fosinopril(Monopril), lisinopril (Prinivil, Zestril), or ramipril (Altace);angiotensin receptor bloekers (ARB), such as azilsartan (Edarbi),eprosaran (Teveten), irbesartan (.Avapro), losartan (Cozaar), olmesartan(Flenicar), and valsartan (Diovan); darbepoetin alfa (Aranesp) orerythropoietin (Procrit, Epogen). As shown in the Examples below, kidneydisease biomarkers include Annexin 5, Nuclear actor of activated cells5and Osmotic stress protein 94.

The following examples are intended to illustrate but not limit theinvention.

Example 1 Collection and Processing of Improved Saliva Samples forSaliva Diagnostics

This experiment provides an exemplary method for collecting andprocessing saliva samples that contain salivary cells. Although asdescribed below, assays based on the collection of human unstimulatedsaliva by spitting or brushing are exemplified, the assay principlescould easily be adapted to protocols that collect and process stimulatedsaliva samples, or use other devices and methods, or collect animalsaliva.

Sample Collection. Unstimulated saliva samples were collected fromhealthy volunteers (5 women, 5 men, 8-53 yrs old). The subjects wereasked to brush teeth and have no food or beverage except water for 30minutes (min) before the collection. Two collection methods were used:(1) Spit was collected into a sterile container such as the 50 ml Falcontube. Subjects spit into the tube several times during about 1.5 minutes(min) until 3 to 6 ml of saliva was collected. During the collection,the tube was kept on ice. (2) A small oral brush described in FIG. 1 wasused to collect saliva by brushing teeth and gum surfaces on both sidesof the mouth for about 20 seconds. When a commercial oral brush (thePROXABRUSH Traveler, Sunstar Americas, Chicago, IL) was used for thiscollection method, the average collected saliva volume was 0.16±0.02 ml.To collect a larger volume of saliva, the brushing was repeated usingadditional brushes. A comparison of saliva samples collected usingself-brushing and brushing performed by an assistant, showed that bothmethods yielded the same average sample volume and cellularcompositiOon.

Sample Quality Tests: (1) Adequate pH (6-8) was assessed by spotting 5microliters of the whole saliva on a pH test strip, or by pressing thetip of the gum brush on a pH strip. Samples tested so far (n>100) hadpH=7.2±0.6. (2) The adequate cell count (epithelial cells andleukocytes) was assessed using one of the following methods. 1. Viablecell count. Saliva sample collected by spitting was mixed thoroughlyusing a sterile 1 ml pipette. Using a sterile pipette tip, 10microliters from the middle of the tube was transferred into a tube with10 microliters of 0.4% trypan blue in the phosphate saline buffer pH7.60 (PBS), mixed using the pipette tip, stained for 10 minutes (min).10 microliters of the mixture was transferred into both chambers of astandard hemocytometer and viable cells (nucleated cells unstained bytrypan) were counted using ×100 magnification. Adequate viable cellcount was at least 0.2×10⁶ cells/ml. 2. Total cell count. Saliva samplecollected by spitting was i:nixed thoroughly using a sterile 1 pipette.Using a sterile pipette tip, 5 microliters from the middle of the tubewas smeared across a small square (about 1 cm²) on a coated microscopyslide (a Silane Prep slide from Sigma, St. Louis, MO). Alternatively,saliva collected by the brush was smeared across about 1 cm length ofthe slide. Air dried saliva smears were fixed. (1 dip in 2% formaline-1%acetic acid 80% ethanol followed by 10 dips in water) and stained usinghematoxylin and eosin (H&E stain, Sigma): 10 dips in hematoxylinfollowed by 10 min in water, 10 dips in 80% ethanol, 10 dip in eosin, 10dips in 95% ethanol, air dry, 2 dips in xylene, coverslip. Cells stainedwith H&E were counted at ×100 magnification. The adequate cell count wasat least 2,500 cells per smear (>5×10⁵ cells /ml). Normal saliva smearstypically contained nucleated epithelial cells and leukocytes as well asabout 10% of epithelial cells without nuclei. (3) The adequate cellularcomposition of the sample was determined based on microscopic inspectionof the H&E saliva smear. A typical saliva sample collected by spittinghad about 60% epithelial cells and 40% leukocytes (monocytes,lymphocytes and granulocytes). Brush-collected samples typically hadabout 50% epithelial cells and 50% leukocytes. In addition, all normalsaliva samples also contained variable amounts of resident bacterial andfungal cells (about 10⁵-10⁷ microbial cells/ml). The salivary microbialcells were associated with the mammalian cells, or ibrmed microbialclumps or were dispersed as single cells.

Salivary Cell Smears on Microscopy Slides (“Smears”). Smears are usefulfor salivary diagnostics in multiple ways. They enable H&E analysis andthe quantitative measurements of cell-associated molecular hiomarkers(proteins, peptides, mRNA, DNA, small molecules such as eicosanoids, orreporters) in salivary epithelial cells, leukocytes and microbial cells.Proteomic markers and small molecules can be measured using theimmunocytochemical staining (“the ICC assay”) or by other assays such asreporter assays or in situ nucleic acid hybridization. Smears wereprepared using following methods. 1. Saliva collected by spitting. Thesample was thoroughly mixed in a tube using a 1 ml sterile pipette.Using a sterile pipette tip, 40 microliter from the middle of the tubewas transferred on a coated slide and the spot was immediately spreadacross the Whole slide using the tip. The tip was tilted at a sharpangle to facilitate the spreading. In some experiments, smaller volumesof saliva were spread in separate areas of the slide to comparedifferent saliva samples on the same slide (e.g., four smears, 10microliter each). 2. Samples collected using oral brush. Immediatelyafier removing from the mouth, the brush was smeared across the lengthof a coated slide. The same brush was used to prepare additional slides(typically, 4 slides per brush). Additional brushes were used to collectmore saliva and prepare a full set of slides for biomarker analysis(e.g., 25 slides were prepared using 7 brushes). Smears prepared singthe spit or brush methods were air dried at room temperature (RT) for atleast 30 min, fixed in 10% normal buffered formalin for 10 min, followedby 3×5 min rinses PBS, 5 min in water and 5 min each in 80%, 95% andabsolute ethanol. Dry fixed slides were stored in a standard histologyslide box at RT. The fixed slides were stable for over 3 years based onthe ICC assay using control antibodies.

Salivary ICC Assay. The main advantages of ICC are sensitivity (specificstaining of single cells corresponding to 0.1-1 pg/ml antigenconcentration) and specificity (each marker stains specific cell typeand has a characteristic cellular localization). To demonstratecompatibility with ICC, representative saliva smears were stained withcontrol antibodies. Positive control antibodies were specific forantigens consistently expressed by salivary epithelial cells orleukocytes. As positive controls, EMA(a membrane antigen on about 30%salivary epithelial cells; mouse IgG2a, 0.2 microgramfml, Biogenex, SanRamon, CA) and CD68 (a cytoplasmic antigen in salivary monocytes, Bcells and neutrophils, mouse IgG1, 0.5 microgramimi, Dako, Carpenteria,CA) were used. Negative control antibodies were mouse monoclonalantibodies (Mab) and rabbit polyclonal antibodies (Pab) specific forirrelevant antigens that are not present in salivary cells. Negativecontrols .matched the concentration, species and type of positivecontrol antibodies and anti-biomarker antibodies. As negative controls,a mouse IgG1 Mab (anti-digoxigenin, 0.5 microgram/ml, Santa CruzBiotechnology, SCBT, Santa Cruz, CA) and a rabbit Pub (anti-Drosophilaarmadillo, 0.2 microgramiml, SCBT) was used. A new protocol wasdeveloped to enable ICC assay of saliva smears. Before staining, dryslides were scored with a diamond pen to outline sections for theapplication of different antibodies (typically 4 control antibodies wereapplied to one slide; in antibody titration experiments, 8 antibodieswere applied to one slide). Assay steps: (1) Based on extensive testing,it was determined that commonly used antigen unmasking methods that useheat treatment (citrate or glycin buffers, 95-100° C. for 10-20 min)were not suitable for saliva smears because >80% cells fell off slidesduring the heat treatment. Therefore, a new method was developed forantigen unmasking in saliva smears: slides were placed in 20 mM citrate,0.1 mM EDTA buffer (pH 3.0) at 37° C. for 60 min, followed by 5 minrinses in water and PBS pH 7.60. (2) Slides were blocked using PBS with7% normal goat serum for 30 min. (3) Based on extensive testing (64rabbit and goat Pabs, 25 mouse and rat Mabs, Mabs), we determined that(i) Mabs (whole culture supernatants, ascites or purifiedimmunoglobulins) and immunoaffinity-purified Pabs were suitable forsalivary immunoassays whereas (ii) Pabs in the form of the whole serum(nonimmune serum or antiserum) or the immunoglobulin fraction of a wholeserum, were unsuitable for salivary immunoassays because they containedantibodies that strongly stained 10-30% salivary microbial cells evenWhen highly diluted. (<10:1000). The anti-microbial affinity of wholeserum has never been reported previously, probably because typicalsamples for trumtmoassays are sterile tissue culture cells, blood cellsand fixed tissues that were stripped of resident microbes. Based on theresults, only Mabs or affinity-purified Pabs were used for all salivaimmunoassays (ICC or other assay forrmats). (3) Optimal concentrationsof primary antibodies (the EMA, CD68, digoxigenin, mouse antibodies)were diluted in PBS, pH 7.60 with 1% bovine serum albumin (BSA) andapplied to individual sections on blocked slides after draining off theblocking solution and dividing the cell smear into fields by wipingbetween the outlined sections using a sharply folded paper tissue. Thetotal antibody volume was 0.3 ml per slide. Afterwards, slides wereplaced in a humidified chamber at 4° C. for 16-20 hrs. (4) After 3×5 minrinse with PBS, a secondary antibody was applied for 90 minutes at RT (abiotinylated. goat antibody against mouse and rabbit IgG , Biogenex,1:20 in PBS-BSA), followed by 3×5 min rinse with PBS, enzymaticconjugate for 30 min at RT (a streptavidine-alkaline phosphataseconjugate, Biogenex,1:20 in PBS-BSA), 3×5 min rinse with TBS (50 mM.Tris, 150 mM NaCl, pH 7.60), chromogen (Fuchsin, Dako), 5 ruin waterrinse, hematoxylin stain for 1 min, 15 min water rinse and two 5 minrinses with 95% ethanol. Air dried slides were rinsed in xylene and.cover-slipped before a microscopic examination at ×100 magnification.(5) The staining intensity was quantified using computerized imageanalysis. 3 representative images were captured in each stained sectionand areas with at least 100 epithelial cells leukocytes were outlined ineach image. The mean optical density (MOD) in the outlined area, and thepercent of the stained area (PA), were determined by, applying a colortile to the image. The same color tile was applied to all images toensure consistent MOD and PA measurements. The staining intensity (SI)was calculated as SI=MOD×PA. The mean SI was calculated for 3 images perstain. To determine the reproducibility of the assays, the mean SI wasmeasured in 12 duplicate stains produced in the same and consecutiveassay runs. The measurements were compared using linear regressionanalysis to calculate 95% confidence interval for the mean ofdifferences. The coefficient of variation (CV) was 9.8%, demonstratingthat the measurement was reproducible. Results of the control stainingshowed critical parameters for the ICC assay: sensitivity 0.1-1 pg/ml(based on the EMA antigen concentration), specificity (no staining withnegative control antibodies), intra- and inter-assay reproducibility(<10% CV for the mean SI measurements).

Saliva Protein Lysates. The lysates enable detecting protein, peptideand small molecule biomarkers present is cell-free saliva and releasedfrom solubilized salivary cells. Lysates can be analyzed using ELISA,protein blots, mass spectrophotometry, chromatography or other types ofassays. As explained, in the TCC assay protocol above, antibodiessuitable for saliva immunoassays are Mabs or innaunoaffinity-puritiedPabs, Lysates were prepared using two methods. (1) Spit-based samples: 1ml of a 2× concentrated lysis buffer (LB) was added per 1 ml saliva; thesample was thoroughly mixed using a sterile 1 ml pipette and kept on icefar 30 min. The final concentrations in the lysate were 1 mM EDTA, 1 mMPMSF, 1 mg/ml N-ethylmaleimide, 0.02 mg/ml ovatrypsin inhibitor, 0.1mg/ml aprotinin, 6 mg/ml 4-aminobenzamidine dichloride and a cocktail ofmammalian phosphatase inhibitors from Sigma diluted in PBS. (2) Salivawas collected using the oral brush (FIG. 1 ) as described above.Immediately after the collection, the brush was removed from the handleand suspended in 2× LB. Four brushes (the PROXABRUSH Traveler, SunstarAmericas) were inixed with 0.3 ml of the. LB in one microcentrifuge tubeby swirling the bruShos in the buffer for 30 seconds. The brush was keptin the buffer on ice for 30 min, and then removed. After lysis,iiisoluble material was removed by centrifugation at 12,000 rpm and thesupernatant was transferred to a new tube and immediately frozen at −80°C.

Saliva ELISA. The ELISA assay complements the ICC assay of biomarkers rnsalivary cells by measuring soluble biomarkers. The main benefits ofELISA are sensitivity to 1 pg/ml biomarker concentrations, consistenthigh throughput and reliable metrics (pg/ml concentration) that clearlyshow the success of clinical diagnostic studies. To demonstratecompatibility with ELISA, we used a multiplexed MultiBead ELISA(Inflammatory Panel, Assay Designs, Ann Arbor, MI) to measure 8 controlproteins (IL-1beta, IL-6, IL-8, IFN-gamma, TNF-alpha) and smallmolecules (eicosanoids PGE2 and TXB2) with known concentrations innormal saliva⁸⁻¹⁰. Calibration curves for the analytes were constructedusing serial dilutions of purified standards first in buffer (PBS-BSA)and then saliva matrix (the saliva protein lysate described above, apool from several subjects). The assay was optimized to reach benchmarkvalues of critical assay parameters: limit of detection at 1-10 pg/ml,linear range 10 pg-10 ng/ml, recovery (assay interferents), specificity(no signal with irrelevant purified antigens), intra- and inter-assay.reproducibility (<10% CV for the mean me.asurements of duplicate samplesin the. same and in consecutive assay runs). The optimized assay wasused to measure the 8 control analytes in normal saliva lysates fromindividual subjects.

Saliva DNA Lysates. These lysates enable measuring DNA released fromsolubilized salivary cells. DNA prepared from the lysates can beanalyzed using PCR, DNA blots or other types of DNA assays. Assays ofsaliva DNA have potentially wide applications in human and animaldiagnostics including pharmacogenomics (individualized testing of drugsafety and efficacy), testing for genetic disorders (diseaseprognostics), paternity and forensics. Although as described below, aprotocol for DNA. preparation is exemplified, the protocol principlescould easily be adapted to protocols that prepare RNA, Saliva DNAlysates were prepared from saliva collected by spitting in a tube or byoral brushing as described above. The objective was to develop a simplemethod that could be used in field conditions using reagents and lysatesthat are stable at RT, and can be later processed in a laboratory toprepare DNA suitable for PCR amplification. Six such methods weredeveloped: (1) 0.1 ml of a 5× concentrated lysis buffer (LB) was addedto 0.5 ml spit in a sterile tube, the sample was thoroughly mixed usinga sterile 1 ml pipette or vigorous shaking, Final concentrations in thelysate were 10 mM. Tris-HCl, 10 mM EDTA, 0.1% sodium dodecyl sulphate(SDS). Fresh concentrated Proteinase K (PK, Qiagen, Valencia, CA) wasadded to 10 microgram/ml final concentration, The lysate was incubatedat 50° C. for 1 hr, boiled for 3 min, 25 microliter of 5M NaCl was addedto 0.2 M final concentration, the sample was mixed with 1 ml absoluteethanol, incubated at RT for 20 min, centrifuged at 14,000 rpm for 10min, the pellet was rinsed with 70% ethanol, air dried and dissolved in50 microliter of 10 mM Tris-1 mM EDTA buffer pH 7,60 (TE). (2) Same asMethod 1 but the LB contained diluted PK and was stored at RT for 2 daysbefore use, (3) Same as Methods 1 or 2 but after boiling, 50 microliterof 5 M iced potassium acetate (pH 4.8) was added, the sample was mixedthoroughly, iced for 30 min, centrifuged at 14,000 rpm for 15 min, thesupernatant was transferred to a new tube, mixed with 1 ml of absoluteethanol, incubated at RT for 20 min, centrifuged at 14,000 rpm for 10min, the pellet was rinsed with 70% ethanol, air dried and dissolved in50 microliter of TE. (4) Same as Methods 1-3 but the lysate wasincubated at RT for 18 hrs instead of at 50° C. for 1 hr. (5) 0.5 ml ofspit was mixed thoroughly with 0.1 ml of a concentrated lysis buffer byvortexing or vigorous shaking. Final concentrations in the lysate were:50mM NaOH, 10 mM EDTA and 0.025% SDS, and the pH was about 12. Thelysate was stored at RT for 8 days without additional mixing. On day 9,the lysate was boiled for 10 min, iced to RT, neutralized to pH 7.8 byadding 5 microliter of 2M Tris-HCl pH 7.0 and 25 microliter of 1M HCl.The neutralized lysate contained 50 mM NaCl. Insoluble material waspelleted by centrifugation at 14,000 rpm for 5 min, the supernatant wastransferred to a new tube, 15 microliter of 5 M NaCl was added to finalconcentration of 0.2 M, the lysate was mixed with 1 ml of absoluteethanol, incubated at RT for 20 min, centrifuged at 14,000 rpm for 10min, the pellet was rinsed with 70% ethanol, air dried and dissolved in50 microliter of TE. (6) Same as Methods 1-5 but saliva was collectedusing an oral brush (FIG. 1 ) as described above. Immediately after thecollection, the brush was removed from the handle and suspended in LB.Four brushes (PROXABRUSH Traveler, Sunstar Americas) were mixed with 0.1ml of 5× LB in one microcentrifuge tube by vortexing or by 10× swirlingthe brushes in the buffer. To estimate the DNA concentration and themolecular weight (MW), 5 microliter each of a DNA standard (HyperLadder1, Bioline, Taunton, MA) and the saliva DNA were analyzed using astandards 0.7% agarose TBE gel with 0.5 microgram/ml ethidium bromide.The average yield per 1 ml saliva was: 3±1 microgram of high MW DNA (>20kbp) for Methods using PK (1-4) and 1.5±0.5 microgram of mediate-low MWDNA (1-20 kbp) for Methods using NaOH (5). To show compatibility withPCR, a 500 by fragment of the human IFN-beta gene was amplified in thedifferent saliva DNA preparations using following primers: 5′ ATG ACCAAC AAG TGT CTC CTC CAA A (SEQ ID NO. 1) and 5′ GTT TCG GAG GTA ACC TGTAAG TCT G (SEQ ID NO. 2), and standard hot-start reaction conditionsusing 1.5 mM MgCl₂, 40 Cycles: 94° C. (45 sec); 60° C. (60 sec); 72° C.(60 sec), then final extension at 72° C. (10 min), The PCR product and aDNA standard were visualized using a standard 2% agarose gel stainedwith ethidium bromide.

Salivary Cells. Live, fixed or permeabilized salivary cells are usefulfor salivary diagnostics by enabling the detection of molecularbiomarkers using flow cytometry (FCM) or immunofluorescence assays. Asexplained in the ICC assay protocol above, antibodies suitable forsaliva immunoassays are Mabs or immunoaffinity-purified fabs. Salivarycells were prepared using the tbllowing procedure: Spit was diluted 1:1with a staining buffer (SR: phosphate buffered saline, pH 7.6, 2% BSA,0.1% azide) and centrifuged at. 300 g for 5 min. Brush-collectedsalivary cells were released into SB (10-30 brushes submerged in 1 mlSB, 5 min on ice on a rocker), and centrifuged as above. The cell pelletwas suspended in a minimal volume of SB (e.g., 0.1 ml), 5 microliterwere removed to perform a cell count, and to determine >90% cellviability using trypan blue exclusion as described above. The averageyield was about 6×⁵ mammalian cells/ml spit and about 3×10 4cells/brush. To demonstrate compatibility with ECM analysis, duplicatesamples of salivary cells were stained with control antibodies using astandard protocol for staining of live cells: The cell suspension wasdiluted to get a final cell concentration of about 10⁶ incubated withanti-Fe receptor antibody (CD32, SCBT, 1 mg/ml, 10 min), divided intostaining samples in microcentrifuge tubes (at least 1×10⁵ cells/sample),centrifuged at 300 g for 5 min at 4° C., resuspended in 0.1 ml with aFITC-labeled primary antibody diluted in SB (1:5 diluted CD68-FITC andnormal mouse IgG₁-FITC, SCBT), mixed and incubated on ice in dark for 30min, rinsed 3× with SB and transferred into a Falcon 2052 tube with 0.4ml SB before FACS analysis.

Example 2 Production of Reference Reagents and Materials for SalivaryDiagnostics

This experiment provides an exe uplaiy method for the production ofnovel reference reagents and materials tier saliva diagnostics byinducing cellular stress in cultured normal salivary cells by in vitrotreatment.

Preparation of Stressed Salivary Cells. Saliva samples (6 ml) weresimultaneously collected from 3 healthy volunteers (1 man and 2 women,19-52 years old) using the spit method from Example 1, The acceptabilityof the samples was immediately evaluated using a pH test and H&E stainas described in Example 1. The samples were combined into a “Normal (N)pool”. A portion the N pool was processed into smears and or lysatesusing protocols from Example 1 (e.g., 3 ml was processed into 75smears). The remaining N pool (15 ml) was divided into 5 cultures: (3 mlsaliva, ˜1×10⁶ viable cells/culture), culture). The cultures weremaintained in sterile Petri dishes (polystyrene, 60 mm×15 mm, Sigma) ina standard cell culture incubator at 37° C. for 18 his without addingculture medium. The cultures contained Whole saliva with all normalsalivary cell types: epithelial cells, monocytes, B and T lymphocytes,granulocytes, fungi and bacteria. Each culture was treated by adifferent environmental stressor: (1) Hypersatinity was induced byadding 150 mM NaCl and incubation for 18 hrs, as previously used forcultured kidney cells¹¹. (2) Oxidative stress was induced by adding0,01% azide and 0.2 .M ethanol and incubation for 18 hrs. (3) Heat shockwas induced by incubation at 44° C. for 2 his followed by incubation at37° C. for 16 hrs. Similar conditions were previously used to heat shockHeLa cells¹². (4) Cold shock was induced by freezing saliva at −80° C.for 2 hrs (3 sterile cryotubes, 1 ml saliva/tube), thawing on ice byadding 1 volume of warm growth medium (RPM with 20% fetal calf serum),transfer into a sterile. Petri dish and incubation at 37° C. for 18 hrs.(5) Desiccation was induced by reducing the culture volume to 1 ml usingprogressive evaporation during 2 hrs, followed by 16 hr incubation atthe same volume. A portion of each treated culture (1 ml from treatments1-3, 2 ml from treatment 4 and 0.3 ml from treatment 5) was processedindividually as “Treated (T1-T5)”. Remaining treated cultures werecombined into a “Stressed (S) pool” (about 10 ml) before processing intosmears on slides. The smears were produced using methods from. Example1.

To determine if the treatments induced cellular stress, 40 SR markerswere measured in smears of N pool, S pool and T1-T5 using the ICC assayprotocol described in Example 1. The primary antibodies were a pool ofantibodies against 40 SRP markers (see Table 2) and control antibodieswere as described in Example 1. The treatment was considered successfulif the average SR marker level was over 3 fold highe saliva (T1-T5, Spool) than in the N pool, see FIG. 2 .

Although stressed salivary cells can be prepared from using one donorand a single environmental stressor, the preferred method describedabove is based on the combination of saliva samples from several donorstreated using 2 or more different environmental stressors. The preferredmethod produces a broad-based cellular stress in saliva, as salivarycells from different genetic backgrounds respond to the variousstressors by activating multiple stress response pathways. Thebroad-based cellular stress results in altered levels of numerousbiomarkers that are affected by cellular stress. The induced biomarkersare present both within salivary cells and also secreted into theculture medium. FIGS. 3A-3H document that treatment of saliva cells bythe preferred method increased levels of SR biomarkers more than 20-foldindicating abroad-based cellular stress. FIG. 4 shows that at least 50individual SR markers were induced by the preferred method indicatingbroad-based activation of the 10 principal SR pathways monitored by the40 markers (see Table 1).

FIG. 5 shows that (i) the SR marker profile induce(by desiccation wasreproducible in salivary cells from different subjects, (ii) SR profilesdiscriminated. between effects of desiccation, heat shock andhypersalinity and (iii) three SR markers were sufficient to discriminatebetween effects of desiccation and heat shock.

Broad-based cellular stress in saliva produced by the preferred methodis directly relevant to clinical salivary diagnostics because a verysimilar broad-based cellular stress was found in saliva samplescollected from subjects with disease or trauma, see FIGS. 3A-3D.

The 40 SR markers were detected in volunteers with inflammatoryconditions that commonly affect the oral cavity (gingivitis andperiodontitis. These volunteers typically had about 5-10% higherconcentration of salivary leukocytes. The average SR markerconcentration was less than 1.1-fold higher in the saliva withinflammatory conditions than in saliva from subjects without thecondition (n=10), which is a statistically insignificant. This resultindicates that salivary diagnostics of disease or trauma is not affectedby common oral inflammatory conditions.

Example 3 Development of Assays for Salivary Biomarkers

This experiment provides an exemplary method for the development of asalivary biomarker assay. The method uses reference reagents andmaterials prepared as described in Example 2. Two types of laboratorysaliva immunoassays are exemplified, the immunocytochemical (ICC) assayand the ELISA assay.

The ICC assay. Methods in Example 2 were used to prepare referenceslides for the assay: salivary cell smears of the N and S pools. Thereference slides were first used to determine the optimal concentrationof the tested anti-marker antibody. The reference slides were preparedusing 20 microliter of the N pool horizontally smeared across the top ofthe slide, and 20 microliter of the S pool smeared across the bottom ofthe slide. Before staining, the slide was vertically divided into 4sections by scoring the opposite side of the slide with a glass pen sothat cacti section contained the N pool on the top and the S pool on thebottom. The sections were stained with 4 serial dilutions of theanti-marker antibody using the ICC staining protocol from Example 1.Parallel slides were stained with the control antibodies described inExample 1. The optimal concentration of the anti-marker antibody wasidentified based on the smallest detectable specific staining in the Npool, and the highest signal ratio between N and S pools. Thesensitivity of the assay was shown based on the detection of singlestained cells. The specificity of the assay was shown as the absence ofstaining with the negative control antibody. The reproducibility of theassay was shown as <10% CV for repeated measurements of the meanstaining intensity in duplicate samples N and S pools in the same assayrun and in 3 consecutive runs. Optimal concentrations of 52 SR markersdetermined using this method are in Table 5.

TABLE 5 Antibodies for the Detection of SR Biomarkers in Salivary CellsANTIGEN ANTIBODY M DF ASK-1 AAP-480 1 1000 Endorphin beta MAB0905 1 100CARD 9 905-188 1 600 Caspase 8 AAP-118 1 50 Cyclin D1 KAM-CC200 1 100Cox-2 sc-7951 2 600 Cytochrome 450 MFO-100 1 600 CYP450 reductaseOSA-300 1 1000 EGFR sc-03 2 150 Ferritin A0133 3 3000 Fos 905-640 1 10Glucocorticoid receptor sc-8992 2 600 GroEL SPS-875 1 600 Grp58 SPA-5801 1000 Grp75 SPA-825 1 50 GSTp A3600 3 1000 HO-1 SPA-895 1 4000 HSF-1SPA-901 1 600 Hsp 25 SPA-801 1 400 Hsp 27 SPA-800 1 100 Hsp 40 SPA-400 1150 Hsp 60 SPA-804 1 750 Hsp 70 SPA-810 1 1000 Hsp 90 SPA-830 1 20 IL-1beta sc-7884 2 800 IL-6 sc-7920 2 1000 IL-8 sc-7922 2 800 IL-10 sc-78882 800 iNOS KAP-NO001 1 30 Jun KAP-TF105 1 150 Laminin PU078-UP 1 150Leptin receptor sc-8391 2 10 Metallothionein MO639 3 15 Mekk-1KAP-SA001E 1 100 Mek-1 KAP-MA010E 1 400 MMP-9 905-486 1 1500 p53KAM-CC002 1 50 PBR sc-20120 2 400 Saliva alpha amylase sc-25562 2 500Serotonin sc-73024 2 10 Serotonin R1A 905-741-100 1 100 Substance Psc-58591 2 100 SOD Cu SOD-100 1 800 SOD EC SOD-106 1 400 SOD Mn SOD-1101 600 TGF beta sc-7892 2 400 VIP sc-20727 2 100 ALR sc-33219 2 250 AQP5sc-28628 2 600 BGT-1 B1082-10 4 100 SAPK KAP-SA011 1 50 p38-MAPKKAP-MA022 1 30 M, Manufacturer: 1-Assay Designs, Ann Arbor, MI. 2-SantaCruz Biotechnology, Santa Cruz, CA. 3-Dako, Carpinteria, CA. 4-USBiological, Swampscott, MA. DF, Dilution Factor.

The ELISA Assay. Methods in Examples 1 and 2 were used to preparereference samples for the assay: protein lysates of the N and S pools.The tested biomarker was analyzed using a commercial ELISA assay usingmethods and the control ELISA assay as described in Example 1. The assaywas optimized to achieve benchmark values for critical assay parametersas described in Example 1. For large marker panels, the development of amultiplexed ELISA assay (e.g., the 8-plex MultiBead ELISA, AssayDesigns) was preferred over the single ELISA assay based on nearly20-fold lower sample volume per analyte and lower cost per sample forthe multiplexed ELISA.

Example 4 Determination of Baseline Concentrations for SalivaryBiomarkers

This experiment provides an exemplary method for measuring baselineconcentrations of saliva biomarkers using two complementary assays, ICCfor cell-associated biomarkers in saliva smears, and ELISA for solublebiomarkers in saliva lysates. The method uses reference reagents andmaterials prepared in vitro as described in Example 2.

Saliva samples (3 ml) were collected from 10 healthy volunteers at 6time points (3 days, 8 am and 3 pm) and used to prepare “individualsmears” and “individual lysates” using methods from Example 1. On thefirst collection day, 1 ml aliquots of each sample were combined (20 ml)and used to prepare smears and protein lysates from N and S pools usingMethods from Examples 1 and 2. Methods from Example 3 were used tovalidate saliva ICC and ELISA assays of the tested biomarker.

Using the validated ICC assay, the tested bioarker was measured intriplicate slides of the individual smears. Smears of the N pool and Spool were used as reference slides with normal and increased levels ofsaliva biomarkers. The mean SI was determined for each smear using imageanalysis method from Example 1.The baseline was calculated as theaverage of the mean SI measurements in the individual samples.Individual and daily variability was determined as the standarddeviation from the baseline.

Using the validated ELISA assay, the tested biomarker was measured induplicate samples of individual lysates. Protein lysates of the N pooland S pool were and used as reference samples with normal and increasedlevels of saliva biomarkers. The baseline was calculated as the averageconcentration in the individual samples. Individual and dailyvariability was determined as the standard deviation from the baseline.

Example 5 Construction of a Biomarker Panel for Salivary Diagnostics

This experiment provides an exemplary method for constructing abiomarker panel that is useful for salivary diagnostics of healthdisorders.

Potential Markers. Potential markers were identified using two methods:(1) Articles describing the molecular mechanism of cellular stressresponses (SR) associated. with health disorders were collected frompeer-reviewed scientific literature. Meta-analysis of the articles wasused to select potential biomarkers based on their association with oneor more universal SR pathways that are activated in different cell typesduring more than one health disorder⁴⁻⁷. Ten universal SR pathways aredescribed in Table 1. Protein lysates of the N pool and S pool wereprepared using methods from Example 2. The lysates were analyzed toidentify differentially expressed proteins and peptides using a methodwith a sufficiently high sensitivity and peptide separation to enablereliable sequencing and identification of peptides in a complex proteinmixture such as the saliva lysate, for example the isotopic labelingcoupled with liquid chromatography tandem muss spectrometry(IL-LC-MS/MS). Potential biomarkers were identified based on more than2-fold difference in the concentration between the S and N pools.

Candidate Marker Panel. Reference slides and protein lysates wereprepared. from the N pool, S pool and treated cultures T1-T5 usingmethods from Examples 1 and 2. Methods from Example 3 were used tovalidate ICC and ELISA immunoassays for potential saliva markers.Methods from Example 4 were used to measure the normal baseline andvariability of the potential markers. Candidate biomarkers were selectedfrom the potential biomarkers using following criteria: (1) Each markerhad a stable baseline in normal saliva based on less than 2-foldindividual and daily differences in the marker concentration. In suchmarkers, the ratio between the baseline concentration and the standarddeviation of the baseline is less than 0.65. (2) The concentration ofeach marker was more than 3-fold different between the S and N pools.Preferred markers had. more than 3-fold increased concentration in the Spool relative to the N pool. (3) When combined into a panel, the markersdiscriminated between the T1-T5 saliva samples. A panel of 52candidatesalivary biomarkers identified using this method is shown inTable 5 and FIGS. 2, 3A-3H, 4 and 5 .

Initial Clinical Validation. A small-scale clinical study was used todemonstrate that a candidate marker panel had a potential diagnosticvalue for a specific medical condition. Clinical saliva samples and goldstandard indicators of the medical condition were collected usingmethods from Example 1. A practical limit for the volume of clinicalsaliva samples was about 3 ml since in many medical conditions patientscannot produce as much saliva as healthy people. The saliva samples wereprocessed into saliva smears and protein lysates using methods fromExample 1. Individual biomarkers were measured in the smears and lysatesusing the validated. ICC and ELIS.A assays. The assays used referenceslides and lysates with normal and increased levels of saliva biomarkers(the N pool and S pool) prepared from. normal saliva using methods fromExamples 1 and 2. The discrimination of the medical condition using thesaliva biomarker panel was determined using correlation analysis withthe gold standard indicator. The diagnostic accuracy of the salivabiomarker for the threshold value of the gold standard indicator wasdetermined using the Receiver Operator Characteristics (ROC) curveanalysis, which provided the criterion values (cutoff sensitivity andspecificity values that divide true negatives and true positives) andthe Area-Under-Curve value (AUC)¹³⁻¹⁵. Optimized biomarker panel wasconstructed by combining a minimal number of markers that classified themedical condition with the greatest AUC value and the most_narrow rangeof criterion values.

Large-Scale Validation. To efficiently measure biomarkers in largesample sets, a multiplexed ELISA assay for the optimized salivabiomarker panel was produced using a commercial assay platform such insuch as MultiBead ELISA (Assay Designs) and methods from Example 3. Theassay used reference lysates with normal and increased levels of salivabiomarkers (the N pool and S pool) prepared from normal saliva usingmethods from Examples 1 and 2. Biomarker measurements obtained by themultiplexed ELISA were used to construct the final biomarker panel thataccurately discriminated, the specific medical condition and was notaffected by potentially contbunding variables such as gender, age andoilier medical conditions.

The final biomarker panel was used for the forward design of acommercial diagnostic test using a mature assay technology with provenacceptability by regulators and customers such as the lateral-flowiiimitmoassay (LFIA)¹⁶⁻¹⁷. The multiplexed ELISA assay was used as thereference assay in the testing of the commercial test. The prototypetest was optimized using reference lysates with normal and increasedlevels of saliva hiomarkers (the N pool and S pool) prepared from normalsaliva using methods from Examples 1 and 2.

Example 6 Saliva Test for Monitoring Hydration Status

A rapid saliva test for ≥3% dehydration has been developed usingfollowing steps: (1) A candidate panel of 52 SR markers (see Table 5)was constructed using methods from Example 5. (2) The initial clinicalvalidation of the panel used methods from Example 5 and a laboratorystudy of dehydration induced in healthy volunteers (n−15) by exercise inheat without fluid intake. The study design discriminated betweeneffects of dehydration and exercise-beat. Stable euhydration before thetrial was documented based on consistent body mass (±1%), plasmaosmolality <290 mOsmol/kg, urine specific gravity <1.02 for 3 days⁵⁶.During the trial, progressive dehydration from 1 to 6% was monitored by1-4% weight loss. Samples of saliva, blood and urine were collected at 9time points. The blood and urine samples were used for standardlaboratory tests of the hydration status including the gold standardtest (plasma osmolality). The clinical saliva samples were collected andprocessed into smears and protein lysates using methods from Example 1.Individual 52 SR markers were measured in the smears and lysates usingoptimized ICC and ELISA protocols as described in Example 5. The assaysused reference slides and lysates with normal and increased levels ofsaliva biomarkers (the N pool and S pool) prepared from normal salivausing methods from Examples 1 and 2. The SR marker measurements werecorrelated with plasma osmolality to determine whether the SR markermeasurements were significantly related to plasma osmolality and notaffected by potentially confounding effects of exercise-heat, gender andsampling variables. The diagnostic accuracy of the SR markers for theplasma osmolality threshold (296 mOsmoL/kg indicating 3% dehydration)was deterrnined using the ROC curve analysis as described in Example 5.A minimal panel of SR markers that had the best diagnostic accuracy for≥3% dehydration was selected using methods from Example 5.

(3) Methods from Example 5 and a large clinical study of dehydration(n=100) were used to construct the optimized SR marker panel. (4) Theoptimized SR marker panel was used to produce a prototype LFIA device.The prototype was optimized using reference saliva lysates as describedin Example 5. The optimized prototype was tested extensively usingclinical saliva samples to demonstrate reliability, accuracy,applicability for field use and regulatory requirements. The multiplexedELISA assay was used as a reference assay for the. LFIA. The LFIA deviceshowed actionable levels of dehydration based on plasma osmolalitythresholds: normal (plasma osmolality ≤290 mOsmol/g), moderatelydehydrated (2-3% dehydration, osmolality 291-296) and severelydehydrated (dehydration >3%, osmolality >296), see FIG. 6 . The benefitof identifying moderate dehydration is that it can be treated in thefield by simple oral rehydration that prevents progression to severedehydration that might require hospitalization and intravenousrehydration.

Example 7 Saliva Test For Monitoring HIV/AIDS Risk and Treatment Outcome

A rapid saliva test for predicting AIDS risk treatment outcome has beendeveloped using following steps: (1) A candidate panel of 52 SR markers(Table 5) was constructed using methods from Example 5. (2) The initialclinical validation of the panel used methods from Example 5 and salivasamples from HIV/AIDS patients (n=100) with CD4 counts >500, 200-500 and<200 cells/mm³. The study subjects had stable CD4 counts during 6inonths before enrollment, and also at when tested during the officevisit when the saliva sample was collected^(68,70-71). The clinicalsaliva samples were collected and processed into smears and proteinlysates using methods from Example 1. The individual 52 SR markers weremeasured in the smears and lysates using optimized ICC and ELISAprotocols as described in Example 5. The assays used reference slidesand lysates with normal and increased levels of saliva biomarkers (the Npool and S pool) prepared from normal saliva using methods from Examples1 and 2. SR marker measurements in the clinical saliva samples werecorrelated with matched CD4 counts (the CD4 count measured during thesame office visit when the saliva was collected). The diagnosticaccuracy of the SR markers for threshold. CD4 counts (≥500 and ≤200cells/mm³) was determined using the ROC curve analysis as described inExample 5. A minimal panel of SR markers with the best diagnosticaccuracy for the threshold CD4 counts was selected using methods fromExample 5.

(3) Saliva samples were collected from HIV patients (n−110) at 5 timepoints during the initial year of the first-line cART. Prior theenrollment, patients had unsuppressed baseline viral load of ≥500copies/ml and a baseline CD4 count <200 cells/mm³. Benchmarks forsuccessful cART outcome after 9 months (expected in 70-90% of thepatients) were: viral load <50 copieslml, CD4 count increased ≥100cells/mm3 above the baseline and no AIDS-defining event or death⁶⁸⁻⁷³.The minimal panel of SR markers produced by the previous study wasmeasured in saliva lysates using multiplexed ELISA as described inExample 5. SR marker measurements were correlated with CD4 count andviral load to determine the prognostic accuracy of SRP markers for cARToutcome. The benchmark for prognostic accuracy was the hazard ratio fromCox proportional hazards models at 95% confidence interval. Thebenchmark for prognostic independence were higher critical chi-squarevalues for Cox models containing SR markers compared to models with CD4count and viral load alone^(69,72). Results of the study were used tooptimize the minimal SR marker panel as outlined in. Example 5.

(4) The optimized minimal SR marker panel was used to produce aprototype LFIA device. The prototype was optimized using referencesaliva lysates as described in Example 5. The optimized prototype wastested extensively using clinical saliva samples to demonstratereliability, accuracy, applicability forr field use and regulatoryrequirements. The multiplexed ELISA assay was used as a reference assayfor the LFIA. The LFIA device showed actionable levels of AIDS riskbased on threshold CD4 counts: low (CD4 count >500 cells/mm³), moderate(CD4 count 500-200 cells/mm³) or high (CD4 count <200 cells). Thebenefit of identifying a moderate AIDS is that it indicates the need forstarting or modifying cART therapy to prevent progression to severe AIDSrisk.

Example 8 SRP Analysis of Post-Traumatic Psychological Stress

Multi-SRP assay of saliva was applied to the study of post-traumaticpsychological stress. Multi-SRP scores were measured in salivary cells(FIG. 8A). The scores strongly correlated with self-reported healthstatus and provided actionable health care information: nonnal dailyactivities were possible at baseline and mildly elevated multi-SRPscores and bed rest was needed at high multi-SRP scores (FIG. 8B).

FIGS. 7A-7B: Multi-SRP scores during post-traumatic psychologicaldistress. Saliva samples were collected from a healthy subject atdifferent time points before and after psychological trauma. FIG. 7A,Multi-SRP staining of saliva cells. Original magnifications: ×200, FIG.7B, The SRP score was calculated as the ratio between the averagestaining intensity across 900 saliva cells, and the maximum stainingintensity value for saliva cells. The staining intensity was quantifiedusing image analysis. Baseline was calculated as the average acrossmulti-SRP scores for six time points before the psychological stress.The error bars are standard deviations. During the psychologicaldistress, multi-SRP scores correlated with the functional state. Normaldaily activities were possible till Day 8 when fatigue was reported.Health status deteriorated on Day 12 and a bed rest was required due todizziness and nausea. Normal health status was reported on Day 45 posttrauma.

Example 9 SRP Biomarkers for Dehydration

The pathway activation index was calculated using a proprietary datamining algorithm and data from the Phase 1 studies. FIG. 8A shows thatthe neuro-endocrine signaling pathway was preferentially upregulated inacute dehydration consistent with early role of systemic hormonalsignalimg in the maintenance of water and sodium homeostasis. FIG. 8Bshows that the pathway signature is different after 12 hrs of persistentdehydration: the dominant pathways are cellular detoxification, osmoticstress response and DNA repair, consistent with adaptive response tocellular and molecular effects of intracellular water loss and increasedsalinity. FIG. 8C shows that dehydration and concussion have differentpathway signatures even though they share some physical symptoms (e.g,lausea, headache), demonstrating specificity for dehydration. Eighty SRPbiomarkers were measured in 195 saliva samples collected from a clinicalstudy of experimentally-induced dehydration. The Pathway activationindex was calculated for Acute 4% hypertonic dehydration, Chronic (12hrs) 4% hypertonic dehydration, Dehydration and concussion using aproprietary algorithm. The results indicate that the signaling pathwayis indicated in acute dehydration and osmotic stress, cellular detox andDNA pathways are indicated in chronic dehydration.

Example 10 Saliva Quality Control

It was discovered that human saliva contains a large number of liveepithelial cells and leukocytes (2×10⁶/ml), and showed that the cells insaliva actively express disease biomarkers. Classical methods for salivacollection (Salivette device, filtered saliva) do not retain wholecells. To take advantage of the diagnostic, potential of the cells, newmethods that reproducibly collect whole saliva including the cells andpreserve molecular integrity of saliva proteins were developed. Salivasamples are aliquoted and stored at −80° C and monitored using a SalivaQuality Control procedure. Optimized algorithm (TripartiteClassification Algorithm, TCA) was developed for accurate and reliablequantification of biomarker signals in INC assays of whole saliva.

The TCA algorithm was applied to validate quantitative. IHC assays usingstandard calibration curves of 5 different biomarkers as illustrated inFIGS. 9A-9B. The IHC assay procedure validated based on high sensitivity(single cells <0.1 pg/ml), reproducibility (mean CV≤20% for duplicatesamples) and accuracy across 40-fold change in biomarker concentration(linear dynamic range, R2≥0.95). The image analysis also allowscalculating the distribution of the biomarker signal between cells andfluid. The biomarker distribution between saliva cells and fluid isconfirmed using Western blot. Knowing where is the biomarker located insaliva is critical for designing the commercial HSM test:cell-associated biomarkers require a lysis step before entering the teststrip, Specifically, reference whole saliva was concentrated 40 fold.Duplicate samples of 16 serial dilutions (1× to 40× concentrated saliva)were spread on slides, and Mucin 1 was detected using sandwichimmunoassay with red Fuschsin label. The red signal volume wasquantified using the TCA algorithm. FIG. 9A. Images of 1× to 40×concentrated saliva stained for Mucin 1 (magnification ×200), FIG. 10BStandard calibration curve for the Mucin1 IHC assay has a linear dynamicrange across 40-fold signal increase.

A immunoblotting method was developed for quantification of proteinbiomarkers in whole saliva: Standardized volume of saliva sample (wholesaliva, 30× concentrated saliva cells or cell-free saliva) and a proteinstandard (recombinant protein) are analyzed using Criterion SDS-PAGEgels. Criterion gels make possible QC monitoring across the workflow:total protein is UV imaged in the gel and on the blot to monitormolecular integrity and transfer efficiency. A specific biomarker isdetected using ECL sandwich immunoassay with a chemiluminescentsubstrate. The chemiluminescent signal is recorded as a series ofexposures (typically 1 s-10 min) using the Chemidoc system (Bio-Rad).Digital image analysis is used to determine the molecular weight,protein concentration and localization of the biomarker in cells and/orfluid, see FIG. 16 . FIG. 16 shows that the saliva WB assay has a highsensitivity (100 ng/ml), reliability (mean CV≤20%) and accuracy across40-fold change in biomarker concentration (R2≥0.95 linear dynamicrange). Specifically, recombinant Hsp27 protein, whole saliva, salivacells and saliva fluid were analyzed using digital Western blot todetermine the MAY, protein concentration and localization of the Hsp27in whole saliva. Calibration curve was constructed using 7 serialdilutions of recombinant Hsp27 (triangles): 100 pg-4 ng/lane (100 ng-4ug/ml) (FIG. 10 ). 4 dilutions of whole saliva (open circles) were usedto determine the Hsp27 protein concentration in the whole saliva.

Example 11 Identificaiton of 20 Candidate Biomarkers for Dehydatton

Existing saliva samples were obtained from clinical and fieldstudies.Two hundred three samples were collected during field studies ofdehydration in US Marines and 195 samples were collected during aclinical study of dehydration. Fifteen healthy men and women age 18-40were enrolled. Each subject was tested for 8 days to establisheuhydrated baseline and daily variability, fbllowed by hypertonicdehydration induced by exercise in heat, euhydrated exercise in heat(control) and isotonic dehydration induced by a diuretic pill (Lasix).The exercise was conducted in an Environmental Chamber with controlledtemperature (86-95° F.) and humidity (20-35%). After each dehydration, astandard protocol was used to produce a full rehydration in 2 hrs, basedon return to baseline body weight and urine specific gravity (USG). Nudebody weight and samples of saliva, blood and urine were collected at 13time points, and used to determine standard hydration indicators: bodymass loss percent (BML%), plasma osmolality (Posm) and USG. In addition,blood and urine were used for clinical laboratory tests: ComprehensiveMetabolic Panel (CMP), Coniplete Blood. Count (CBC) and Urine Analysis(UA) that were reviewed by licensed MD to ensure subject health andsafety. Eighty SRP biomarkers were measured in the 398 existing salivasamples from the clinical and field studies using the quantitativedigital IHC assay described above. Thirty five biomarkers specific fordehydration were identified based on two critical parameters: 23-foldincrease in dehydration (clinical and field), and <2-fold increase ineuhydrated controls (clinical and field). The field saliva samplesprovided a critical refinement by showing which specific biomarkers werenot confounded by severe dehydration >4% (USG>1.03, N=36 field samples),extreme environments (110° F., 10% humidity), sleep deprivation,operational stress or tobacco use. These potentially confoundingconditions could not be tested in a clinical trial. Diagnostic accuracyoften specific biomarkers was determined using ROC curve analysis. Bestbiomarkers were selected based on diagnostic accuracy 280% fordehydration. Twenty candidate biomarkers are listed in FIG. 11 .

Example 12 Feasibility Trial

Candidate biomarkers of dehydration were measured in 560 saliva samples.These saliva samples were collected from different subjects than theprevious examples and therefore provide an independent validation. Thebiomarkers were measured using the quantitative digital THC and Westernblot assays described previously. FIGS. 12A-12B show the fold increasein normalized bioinarker levels d.uring dehydration relative toeuhydrated baseline. EUH, euhydrated baseline. HYP, hypertonicdehydration. ISO, isotonic dehydration. Error bars represent standarderror. Diagnostic accuracy of the individual biomarkers was determinedusing ROC curve analysis of the individual IHC data (not shown). The EECdata correlated with the Western blot data based on Spearman'scorrelation coefficient Rho. Ten biomarkers were validated based ondiagnostic accuracy 280% for dehydration. The 10 validated biomarkersare listed are acid trehalase-like protein, aldose reductase, aquaporin5, induced nitric oxide synthase 2, mucin 1, neuropathy target esterase,nuclear factor of activated T cells 5, osmotic stress protein 94,sodium/myo-inositol cotransporter and trehalase. Each validatedbiomarker has a diagnostic accuracy 280% for at least 2 dehydrationstates and rehydration, and diagnostic accuracy <65% for euhydratedexercise demonstrating that the markers are not confounded by thecontrol. The diagnostic accuracy of the 10 biomarkers is notsignificantly of by gender, daily variability or diurnal variability(data not shown).

Example 13 Analysis Of Biomarkers for Dehydration

Stepwise Logistic Regression and Multivariate ROC curves (SAS DAP Pro11) were used to select a minimal panel of biomarkers with bestindependent predictive value and highest diagnostic accuracy. Top 3biomarkers are Acidic Trehalase-like protein 1 (AIHL), Osmotic stressprotein 94 (OSP94) and Sodium: myo-inositol cotransporter (SMIT), Thestatistical analysis was based on results of two orthogonal biomarkerassays, MC and Western blot, robustly correlated based on Spearman'scorrelation coefficient Rho=84-90% (data not shown). To determine thediagnostic accuracy, specificity, sensitivity and cutoff value, a Panelscore has been defined as a single numerical value representing all 3dehydration biomarkers. Algorithm for calculating the panel score P fromthe normalized biomarker data is provided below:

Panel score P=κ1χ1+κ2χ2+κ3χ3

Whereas the values of χ are hiomarker scores, for example χ1 is ATHLscore, χ2 is OSP94 score, χ3 is SMIT score and the values of κ areconstants, for example κ1=1.0, κ2=20 and κ3=10.

The Panel scores were used for ROC analysis. The diagnostic accuracy,specificity and sensitivity of the final hiomarker panel are shown inFIGS. 13A-13C, and demonstrates that the diagnostic accuracy is notconfounded by the type of level of dehydration, effects of gender, dailyand diurnal variability or euhydrated exercise (control). Table 15 alsoshows that the biomarker panel accurately detects rehydrationimmediately after subjects completed fluid replacement. Specificity ofthe biomarkers was demonstrated based on correlation with standardhydration indicators, and correlation between blomarkers and thehydration level (euhydrationidehydration) (FIGS. 14A-14D). Specifically,Panel score was correlated with measurements of Body Mass Loss (BML%),Plasma Sodium (Sodium), Plasma Osmolality (Posm) and Urine SpecificGravity (USG). E, euhydration, D, dehydration, R, rehydration,

The minimal set of bioniarkers of dehydration have the tbilowingcharacteristics:

-   -   1) best predictors independently associated with dehydration    -   2) 94% diagnostic accuracy, 88% specificity and 88% sensitivity    -   3) biomarker scores correlate with standard indicators Body Mass        Loss, Plasma Osmolality, Plasma sodium and Urine Specific        Gravity    -   4) diagnostic accuracy is not confounded by type of dehydration        (hypertonicfisotortie); level of dehydration (2% and 4%): timing        of dehydration (acute or 12 hrs); rehydration; euhydrated        exercise; heat; gender; daily and diurnal variability and field        condition, sleep deprivation and tobacco use    -   5) confirmed by IHC, Western blot and Mass spectroscopy.

Example 14 Pathway Signature for HIV

SRP biamarkers were measured in 58 saliva samples from patients withART-suppressed or unsuppressed HEY, acute HIV and HIV negative STD(syphilis, gonorrhea or chlamydia) (FIGS. 15A-15C). Pathway activationindex was calculated from biomarker data using a proprietary algorithmdescribed previously. The adhesion, cytoskeleton intdexosortle pathwaywas preferentially upregulated in unsuppressed HIV, consistent withcytoskeletal stress due to high HIV virus production. Multiple pathwayswere moderately upregulated in suppressed HIV, in particular apoptosisand autophagy, consistent with restored immunity and active cellularstress responses due to successful ART therapy. Low stress responseactivation was found in acute HIV consistent with low cytokine levelsreported in post viremic HIV. STD had a different pathway signature thanHIV, demonstrating the specificity of the HIV pathway signature.

Example 15 Identification of HIV Biomarkers

Whole saliva was used to take advantage of cell-associated. HIV whichwas identified as the main source of oral HIV. Current methods usefiltered saliva Which might have resulted in low HIV concentration.Whole saliva samples (98) (3 ml) were collected using a standardizedprocedure. Saliva samples were aliquoted and stored at −80° C., andtested before biomarker assays using a standard QC matrix including thetotal saliva volume, appearance, color, cellular/molecular preservationin epithelial cells and leukocytes. Ninety live SRP biomarkers werequantitatively measured in 89 saliva samples from using high throughputdigital IHC assay. The IHC results were confirmed using the digitalWestern blot. Results identified biomarkers with a significant (P<0.05),over 2-fold change in HIV patients compared to healthy controls.Unsuppressed HIV/AIDS had more altered biomarkers (N=27) then suppressedHIV (N=17) and acute HIV (N=13). HIV specificity was analyzed usingWilcoxon rank sum test (2-tailed test, alpha 0.05). Biomarker profilesin HIV infection (Cohorts 1-3) were significantly different fromprofiles in HIV-negative STD-positive individuals (Cohort 4), P<0.01,This result agrees with. HIV/STD differences demonstrated using thepathway signatures, see FIGS. 15A-15C. To identify candidate biomarkersfor diagnostics of unsuppressed and acute HIV, 39 biomarkers withdifferential expression between Cohorts1/2 and Cohorts 3/4 wereexamined. Diagnostic accuracy of SRP biomarkers was determined usingReceiver Operating Characteristic (ROC) curves. ROC curves wereconstructed fix 39 biomarkers with differential expression (≥2-foldchange, P<0.05) in suppressed/unsuppressed HIV and acute HIV/STD. Thearea-under the-curve (AUC) value was used to determine the diagnosticaccuracy for individual biomarkers. Riomarkers with AUC≥0.8 are in FIG.16 . These markers were further analyzed by multivariate ROC analysis(JMP11Pro SAS) to select minimal biomarker panels with best predictivevalue. The down-selected 4 markers are BST2, salivary agglutinin gp340(SAG), cytoplasmic cytochrome c, and vascular endothelial growth factorC (VEGF-C). All the markers have known roles in host response to HIV.BST2 is a cellular HIV restriction factor, cytoplasmic cytochrome c is amitochondrial protein that triggers apoptosis when released intocytoplasm in HIV-infected cells and is toxic to uninfected bystandercells, SAG binds to the HIV envelope protein gp120 and specificallyinhibits HIV-1 infectivity, VEGF-C is a growth factor upregulated by theHIV Tat-1 protein.

Example 16 Pathway Signature and Biomarkers Specific for NCDT

A full panel of 100 SRP biomarkers was profiled in pooled saliva samplesfrom Neuro-Cognitive Disorder due to TB1 (NCDT) and other diseases.Normalized Pathway activation index (0-10) was calculated from biomarkerdata using a patented algorithm. The arrow indicates the top activatedpathway in each disease. SRP pathways: 1—Oxidative stress,2—Detoxification, 3—Chaperoning, 4—DNA, 5—Adhesion/Cytoskelet n, 6—Cellcycle, 7—Apoptosis, 8—Signaling, 9—Immunity, 10—Microbionie (FIG. 17A).Oxidative stress, cellular detoxification and cytoskeletal damage(Pathways 1, 2 and 5) were preferentially activated in acute TBI (FIG.17B). Pathways 2 and 5 remain highly activated in NCDT but the dominantpathway 7 is apoptosis (FIG. 17F). The pathway signatures based onsaliva SRP biomarkers are consistent with TBI literature. Oxidativestress, cellular detoxification and cytoskeletal damage are known toplay key roles in both primary and secondary injury following acute TBI.Apoptosis is rare in acute mTBI (mild traumatic brain injury,concussion); however, it plays important role in long-term evolution ofneuro-cognitive deficits and neurodegeneration following mTBI. FIGS.17A-17I demonstrate that NCDT has a specific pathway signature, distinctfront other chronic disease states. FIGS. 171-17I also show that acuteand chronic phases of disease pathogenesis have a distinct molecularmechanism not only in TBI but also other diseases.

Using the above described methods, eight candidate NCDT biomarkers wereidentified among 91 SRP biomarkers: Adrenocorticotropic hormone (ACTH),Cytochrome P450 Reductase (CYPOR), Epidermal growth factor receptor(EGFR), Glucocorticoid receptor (GR), Home oxygenase 1 (HO), MAP kinase.Mek-1 (MEK) Natriuretic peptide receptor A (NPR) and Oxytocin receptor(OTR). These biomarkers have diagnostic accuracy ≥80% for NCDT (ROCanalysis), arc≥3-fold increased in NCDT compared to healthy controls,and <2-fold increased in acute TBI and diseased controls (specificity).Stepwise Logistic Regression and Multivariate ROC curves (SAS JMP Pro11) were used to select a minimal panel of biomarkers with bestindependent. predictive value and highest diagnostic accuracy. Top 3biomarkers are Cytochrome P450 Reductase (CYPOR), Natriuretic peptidereceptor (NPR) and Oxytocin receptor (OTR),

CYPOR—The NADPH-cytochrome P450 reductase is oxidative enzyme thatmediates removal of xenobiotics. Cellular detoxification is critical inTBI because increased levels of xenobiotics are generated by oxidativestress and eytoskeletal damage an acute and delayed phases. Alteredexpression of detoxification enzymes in the brain was linked to variousneurological diseases, and overexpression of detoxification enzymesconferred neuroprotection in animal models. These observations suggestimportant role fbr detoxification enzymes such as CYPOR in NCDTpathogenesis.

NPR—The natriuretic peptide receptor A mediates effects of natriureticpeptides ANP and BNP secreted by the heart and the brain. Natriureticpeptides have vasodilating, natriuretic and diuretic activities thatmodulate blood pressure and cerebral blood flow, and can preventhypertension and brain edema. Hypertension and brain swelling are commonin TBI, and BNP plasma concentrations were found continuously elevatedin TBI patients with poor outcomes and cerebral salt wasting. Recently,elevated BNP was linked with deficits in neurocognitive function:memory, processing speed, executive functioning and depressive symptoms,independent of cardiovascular risk factors and cardiac output. Thesefindings suggest—that natriuretic peptides and NPR might play a role inNCDT pathogenesis.

OTR—The oxytocin receptor (OTR) regulates effects of the neuropeptideoxytocin (OT). OT is a systemic hormone and neuromodulator that plays acritical role in social and emotional behavior through reduced anxiety,tear and stress reactivity. Intranasal OT is currently tested as apharmacological agent for the prevention and treatment of PTSD becauseof its anxiolytic and p:rosocial properties. Anxiety disorders andantisocial behaviors (irritability, impulsivity and aggression) are corefeatures of NCDT, suggesting that oxytocin and. OTR could be involved inthe mechanism of NCDT.

Example 17 Pathway Signature and Biomarkers for Heart Failure and KidneyDisease

A full panel of 100 SRP biomarkers was profiled in pooled saliva samplesfrom acute heart failure (AHF) patients stratified into severaldifferent disease phenotypes: Preserved Ejection Fraction (HF-pEF).Restricted Ejection Fraction (HF-rEF), Atrial Fibrillation (AFIB),Chronic Kidney Disease (CKD) and Acute Kidney Injury (AKI). NormalizedPathway activation index (0-10) was calculated from bioniarker datausing a patented algorithm. The arrow indicates the top activatedpathway in each disease. SRP pathways: 1—Oxidative stress,2—Detoxification, 3—Chaperoning, 4—DNA, 5—Adhesion/Cytoskeleton, 6—Cellcycle, 7—Apoptosis, 9—Immunity, 10—Microbiome (FIGS. 17A-17I). Celladhesion/Cytoskeletal stress pathway, apoptosis and oxidative stress(pathways 5, 7 and 1) were preferentially activated both the H.F-pEF andHF-rEF phenotypes (FIGS. 17D and 17H); however, pathway activation wasstronger in HF-pEF than HF-rEF. FIGS. 17A-17I demonstrate that AHF has aspecific pathway signature, distinct from other chronic diseases. FIG.17A-17I also show that the HF-pEF and HF-rEF phenotypes have a distinctmolecular mechanism.

Selected biomarkers were measured in individual AHF patients andcontrols. Normalized data was analyzed using ROC curve analysis todetermine diagnostic accuracy of the saliva biomarkers. ROC analysisresults are provided in FIGS. 19A-19B. Candidate biomarkers wereselected based on highest diagnostic accuracy. AHF and AFIB biomarkersinclude Annexin 5, Cox-2, EGFR, Leptin, MEK1, mTOR, NFAT5, OSP94, SAPKand SOD3. Candidate AKI and CKD biomarkers include Annexin 5, EGFR,Leptin, MEK1, NFAT5 and OSP94.

Example 18 Path AY Signature and Biomarkers for Concussion (Acute mTBI)

A full panel of 100 SRP biomarkers was profiled in pooled saliva samplesfrom acute mTBI (concussion) and healthy volunteers. Normalized Pathwayactivation index (0-10) was calculated from biomarker data using apatented algorithm. The arrow indicates the top activated pathway ineach disease. SRP pathways: 1—Oxidative stress, 2—Detoxification,3—Chaperoning, 4—DNA, 5—Adhesion Cytoskeleton, cycle, 7—Apoptosis,8—Signaling, 9—Immunity, 10—Microbiome (FIGS. 17A-17I). Oxidativestress, cellular detoxification and cytoskeletal damage (Pathways 1, 2and 5) were preferentially activated in acute mTBI (FIG. 17B). FIGS.17A-17I demonstrate that acute mTBI has a specific pathway signature,distinct from chronic TBI (NCDT) and other chronic disease states. FIGS.17A-17I also show that acute and chronic phases of disease pathogenesishave a distinct molecular mechanism not only in TRI but also otherdiseases. Figures 20A-20B show results of two independent studies ofmTBI patients. Both studies showed strong increase in over 50 SRPbiomarkers in whole saliva. Many of the biomarkers wore consistentlyincreased in both studies demonstrating that the effect is reproducible,

publications and patents discussed herein are provided solely for theirdisclosure prior to the tiling date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed,

All the publications cited are incorporated herein by reference in theirentireties, including all published patents, patent applications,literature references, as well as those publications that have beenincorporated in those published documents. However, to the extent thatany publication incorporated herein by reference refers to informationto be published, applicants do not admit that any such informationpublished after the filing date of this application to be prior art.

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the inventions herein disclosedcan be resorted by those skilled in the art, and that such modificationsand variations are considered to be within the scope of the inventionsdisclosed herein. The inventions have been described broadly andgenerically herein. Each of the narrower species and subgenericgroupings falling within the scope of the generic disclosure also formpart of these inventions. This includes the generic description of eachinvention with a proviso or negative limitation removing any subjectmatter from the genus, regardless of whether or not the excisedmaterials specifically resided therein. In addition, where features oraspects of an invention are described in terms of the Markush group,those schooled in the art will recognize that the invention is alsothereby described in terms of any individual member or subgroup ofmembers of the Markush group.

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Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A method for detecting traumatic brain injury(TBI) in a subject comprising: (i) processing a salivary cell samplefrom the subject for biomarker analysis comprising: (a) applying asample of saliva or salivary cells from a subject to a substrate; (b)fixing the cells: (c) incubating the cells in a citrate buffer at aboutpH 3.0 at 37° C.; (d) contacting the cells with serum; (e) applying aprimary antibody for each of biomarker of a biomarker panel; and (f)detecting the binding of the primary antibody using a secondary antibodyhaving a detectable label, wherein the label is detected optically usinga computerized image analysis, and (ii) detecting an altered level of atleast one biomarker associated with TRI in the sample as compared to acorresponding sample from a subject not having TBI, wherein analteration in the level of biomarker is indicative of a stress responseassociated with TEl, thereby detecting TBI in the subject.
 2. The methodof claim 1, wherein the at least one biamarker is selected from thegroup consisting of ALG-2 interacting protein X (Alix), CD63 (CD63,Tetraspanin, LAMP-3), Cytochrome P450 Reductase (CYPOR), Glucoseregulated protein 58 (Grp58), Hypoxia-induced factor alpha 1 (HIF1),Leptin, MAP kinase Mek-1, mitogen activated (MEK1), MAP kinase Mekk-1,stress activated (MEKK1), Matrix metalloproteinase 9 (MMP9), Natriureticpeptide receptor A (NPR), Neuropathy target esterase (NTE), Nitric oxidesynthase, neuronal nNOS (NOS1), Nitric oxide synthase, Oxytocin receptor(OTR), Superoxide dismutase 3 Extracellular (SOD3), Toll-like receptor 3(TLR3), Toll-like receptor 7 (TLR7), Toll-like receptor 8 (TLR8), or acombination thereof.
 3. The method of claim 1, wherein traumatic braininjury is selected from the group consisting of mild TBI (mTBI,concussion), severe TBI (sTBI) and neurocognitive disorder (NCD) due toTBI (NCDT, DSM-5).
 4. The method of claim 1, wherein the at least onebiomarker is selected from the group consisting of: Cytochrome P450Reductase (CYPOR), Heme oxygenase 1 (HO), MAP kinase Mek-1 (MEK),Natriuretic peptide receptor A (NPR) and Oxytocin receptor (OTR) and acombination thereof.
 5. The method of claim 1, wherein the at least onebiomarker is selected from the group consisting of NTE, HIF-1A, CYPORand OTR.
 6. A method for detecting mild TBI (concussion) in a subjectcomprising: (i) processing a salivary cell sample from the subject forbion arker analysis; and (ii) detecting an altered level of at least onebiomarker associated with concussion in the sample, as compared to acorresponding sample from a subject not having a concussion, wherein analteration in the level of biomarker is indicative of a stress responseassociated with concussion, thereby detecting concussion in the subject.7. The method of claim 7, wherein processing a salivary cell sample fromthe subject for biomarker analysis comprises: (a) applying a sample ofsaliva or salivary cells from a subject to a substrate; (b) fixing thecells; (c) incubating the cells in a citrate buffer at about pH 3.0 at37° C.; (d) contacting the cells with serum; (e) applying a primaryantibody for each of biomarker of a biomarker panel; and (f) detectingthe binding of the primary antibody using a secondary antibody having adetectable label, wherein the label is detected optically using acomputerized image analysis.
 8. A method for detecting concussion in asubject comprising: (i) processing a salivary cell sample from thesubject for biomarker analysis comprising: (a) applying a sample ofsaliva or salivary cells from a subject to a substrate; (b) fixing thecells; (c) incubating the cells in a citrate buffer at about pH 3.0 at37° C.; (d) contacting the cells with serum; (e) applying a primaryantibody for each of biomarker of a biomarker panel; and (f) detectingthe binding of the primary antibody using a secondary antibody having adetectable label, wherein the label is detected optically using acomputerized image analysis, and (ii) detecting an altered level of atleast one biomarker associated with concussion in the sample comprisingsalivary cells from the subject, as compared to a corresponding samplefrom a normal subject, wherein an alteration in the level of biomarkeris indicative of a stress response associated with concussion, therebydetecting concussion in the subject.